Methods and compositions for transducing and expanding lymphocytes and regulating the activity thereof

ABSTRACT

The present disclosure provides methods for genetically modifying lymphocytes and methods for performing adoptive cellular therapy that include transducing T cells and/or NK cells. The methods can include inhibitory RNA molecule(s) and/or engineered signaling polypeptides that can include a lymphoproliferative element, and/or a chimeric antigen receptor (CAR), for example a microenvironment restricted biologic CAR (MRB-CAR). Additional elements of such engineered signaling polypeptides are provided herein, such as those that drive proliferation and regulatory elements therefor, as well as replication incompetent recombinant retroviral particles and packaging cell lines and methods of making the same. Numerous elements and methods for regulating transduced and/or genetically modified T cells and/or NK cells are provided, such as, for example, those including riboswitches, MRB-CARs, recognition domains, and/or pH-modulating agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.16/490,201, filed Aug. 30, 2019; U.S. application Ser. No. 16/490,201 isa 371 of International Application No. PCT/US2018/020818 filed Mar. 3,2018; International Application No. PCT/US2018/020818 is acontinuation-in-part of International Application No. PCT/US2017/023112filed Mar. 19, 2017; a continuation-in-part of International ApplicationNo. PCT/US2017/041277 filed Jul. 8, 2017; a continuation-in-part of U.S.application Ser. No. 15/462,855 filed Mar. 19, 2017, now U.S. Pat. No.10,596,274; and a continuation-of-part of U.S. application Ser. No.15/644,778 filed Jul. 8, 2017, now U.S. Pat. No. 11,111,505; and claimsthe benefit of U.S. Provisional Application No. 62/467,039 filed Mar. 3,2017; U.S. Provisional Application No. 62/560,176 filed Sep. 18, 2017;U.S. Provisional Application No. 62/564,253 filed Sep. 27, 2017; andU.S. Provisional Application No. 62/564,991 filed Sep. 28, 2017;International Application No. PCT/US2017/023112 claims the benefit ofU.S. Provisional Application No. 62/390,093, filed Mar. 19, 2016; U.S.Provisional Application No. 62/360,041, filed Jul. 8, 2016; and U.S.Provisional Application No. 62/467,039, filed Mar. 3, 2017;International Application No. PCT/US2017/041277 is acontinuation-in-part of International Application No. PCT/US2017/023112,filed Mar. 19, 2017; and is a continuation-in-part of U.S. patentapplication Ser. No. 15/462,855, filed Mar. 19, 2017; and claims benefitof U.S. Provisional Application No. 62/360,041, filed Jul. 8, 2016; andU.S. Provisional Application No. 62/467,039, filed Mar. 3, 2017; U.S.application Ser. No. 15/462,855, claims the benefit of U.S. ProvisionalApplication No. 62/390,093, filed Mar. 19, 2016; U.S. ProvisionalApplication No. 62/360,041, filed Jul. 8, 2016; and U.S. ProvisionalApplication No. 62/467,039, filed Mar. 3, 2017; and U.S. applicationSer. No. 15/644,778 is a continuation-in-part of InternationalApplication No. PCT/US2017/023112, filed Mar. 19, 2017, and acontinuation-in-part of U.S. patent application Ser. No. 15/462,855,filed Mar. 19, 2017, and claims the benefit of U.S. ProvisionalApplication No. 62/360,041, filed Jul. 8, 2016, and U.S. ProvisionalApplication No. 62/467,039, filed Mar. 3, 2017. These applications areincorporated by reference herein in their entirety.

SEQUENCE LISTING

This application hereby incorporates by reference the material of theelectronic Sequencing Listing filed concurrently herewith. The materialsin the electronic Sequence Listing is submitted as an Extensible MarkupLanguage (.xml) file entitled “F1_001_US_03CIP1_Sequence_Listing.xml”created on Jul. 28, 2023, which has a file size of 725 KB, and is hereinincorporated by reference in its entirety.

FIELD OF INVENTION

This disclosure relates to the field of immunology, or morespecifically, to the genetic modification of T lymphocytes or otherimmune cells, and methods of making replication incompetent recombinantretroviral particles and controlling the expression of genes therein.

BACKGROUND OF THE DISCLOSURE

Lymphocytes isolated from a subject (e.g. patient) can be activated invitro and genetically modified to express synthetic proteins that enableredirected engagement with other cells and environments based upon thegenetic programs incorporated. An example of such a synthetic protein isa chimeric antigen receptor (CAR). One CAR that is currently used is afusion of an extracellular recognition domain (e.g., an antigen-bindingdomain), a transmembrane domain, and one or more intracellular signalingdomains encoded by a replication incompetent recombinant retrovirus.

While recombinant retroviruses have shown efficacy in infectingnon-dividing cells, resting CD4 and CD8 lymphocytes are refractory togenetic transduction by these vectors. To overcome this difficulty,these cells are typically activated in vitro using stimulation reagentsbefore genetic modification with the CAR gene vector can occur.Following stimulation and transduction, the genetically modified cellsare expanded in vitro and subsequently reintroduced into alymphodepleted patient. Upon antigen engagement in vivo, theintracellular signaling portion of the CAR can initiate anactivation-related response in an immune cell and release of cytolyticmolecules to induce tumor cell death.

Such current methods require extensive manipulation and manufacturing ofproliferating T cells outside the body prior to their reinfusion intothe patient, as well as lymphodepleting chemotherapy to free cytokinesand deplete competing receptors to facilitate T cell engraftment. SuchCAR therapies further cannot be controlled for propagation rate in vivoonce introduced into the body, nor safely directed towards targets thatare also expressed outside the tumor. As a result, CAR therapies todayare typically infused from cells expanded ex vivo from 12 to 28 daysusing doses from 1×10⁵ to 1×10⁸ cells/kg and are directed towardstargets, for example tumor targets, for which off tumor on targettoxicity is generally acceptable. These relatively long ex vivoexpansion times create issues of cell viability and sterility, as wellas sample identity in addition to challenges of scalability. Thus, thereare significant needs for a safer, more effective scalable T cell or NKcell therapy.

Since our understanding of processes that drive transduction,proliferation and survival of lymphocytes is central to variouspotential commercial uses that involve immunological processes, there isa need for improved methods and compositions for studying lymphocytes.For example, it would be helpful to identify methods and compositionsthat can be used to better characterize and understand how lymphocytescan be genetically modified and the factors that influence theirsurvival and proliferation. Furthermore, it would be helpful to identifycompositions that drive lymphocyte proliferation and survival. Suchcompositions could be used to study the regulation of such processes. Inaddition to methods and compositions for studying lymphocytes, there isa need for improved viral packaging cell lines and methods of making andusing the same. For example, such cell lines and methods would be usefulin analyzing different components of recombinant viruses, such asrecombinant retroviral particles, and for methods that use packagingcells lines for the production of recombinant retroviral particles.

SUMMARY

Provided herein are methods, compositions, and kits that help overcomeissues related to the effectiveness and safety of methods fortransducing and/or genetically modifying lymphocytes such as T cellsand/or NK cells. Certain embodiments of such methods are useful forperforming adoptive cell therapy with these cells. Accordingly, in someaspects, provided herein are methods, compositions, and kits forgenetically modifying and/or transducing lymphocytes, especially T celland/or NK cells, and/or for regulating the activity of transduced and/orgenetically modified T cells and/or NK cells. Such methods,compositions, and kits provide improved efficacy and safety over currenttechnologies, especially with respect to T cells and/or NK cells thatexpress chimeric antigen receptors (CARs), and in illustrativeembodiments microenvironment restricted biologic CARs. Transduced and/orgenetically modified T cells and/or NK cells that are produced by and/orused in methods provided herein, include functionality and combinationsof functionality, in illustrative embodiments delivered from retroviral(e.g. lentiviral) genomes via retroviral (e.g. lentiviral) particles,that provide improved features for such cells and for methods thatutilize such cells, such as research methods, commercial productionmethods, and adoptive cellular therapy. For example, such cells can beproduced in less time ex vivo, and that have improved growth propertiesthat can be better regulated.

Provided herein in some aspects are regulatory elements for regulatingthe expression of CARs, mRNA, inhibitory RNA(s), and/orlymphoproliferative elements, for example chimeric lymphoproliferativeelements, in lymphocytes such as B cells, T cells and NK cells.Furthermore, provided herein in some aspects are recombinantretroviruses that express various functional elements and that carryvarious functional elements on their surface, and methods and packagingcell lines for producing the recombinant retroviruses. These recombinantretroviruses and methods and cells for producing the same, overcomeprior art limitations with respect to the number and size in a genome,of different functional elements that provide benefits when deliveredinto a T cell and/or NK cells.

In some aspects, methods are provided for transducing and/or geneticallymodifying lymphocytes such as T cells and/or NK cells, and inillustrative embodiments, ex vivo methods for transducing and/orgenetically modifying resting T cells and/or NK cells. Some of theseaspects can be performed much more quickly than previous methods, whichcan facilitate more efficient research, more effective commercialproduction, and improved methods of patient care. Furthermore, providedherein are methods that in some embodiments utilize recombinantretroviruses provided herein in some aspects along with pharmacologicagents, to provide improved safety mechanisms to help modulate theactivity of transduced and/or genetically modified lymphocytes such as Tcells and/or NK cells. Such methods, compositions, and kits can be usedas research tools, in commercial production, and in adoptive cellulartherapy with transduced and/or genetically modified T cells and/or NKcells expressing a CAR.

Further details regarding aspects and embodiments of the presentdisclosure are provided throughout this patent application. Sections andsection headers are not intended to limit combinations of methods,compositions, and kits or functional elements therein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of illustrative compositions including apackaging cell (100) and a replication incompetent recombinantretroviral particle (200) of one exemplary, non-limiting embodiment ofthe present disclosure, produced by the packaging cell (100). In FIG. 1, various vectors (referred to as recombinant polynucleotides (110))capable of encoding aspects of the invention are packaged into arecombinant retroviral particle (200) that includes in its genome afirst engineered signaling polypeptide that includes one or morelymphoproliferative elements and in some embodiments, a secondengineered signaling polypeptide that is a chimeric antigen receptor, ora CAR. The replication incompetent recombinant retroviral particleexpresses on its membrane, a pseudotyping element (in a non-limitingembodiment, a Measles Virus hemagglutinin (H) polypeptide and a MeaslesVirus fusion (F) polypeptide, or cytoplasmic domain deletion variantsthereof) (240) that allows the replication incompetent recombinantretroviral particle to bind to and fuse with a target cell; anactivation element (in non-limiting embodiments an activation elementthat has a polypeptide capable of binding to CD28 and a polypeptidecapable of binding to CD3) (210 and 220, respectively) that is capableof binding to and activating a resting T cell; and a membrane-boundcytokine (in a non-limiting embodiment, an IL-7 DAF fusion polypeptide)(230). Parts labeled as (250), (260), (270), (280), and (290) are theSrc-FLAG-Vpx, HIV gag matrix, HIV gag capsid, RNA, and HIV pol,respectively.

FIG. 2 shows a schematic of illustrative compositions including areplication incompetent recombinant retroviral particle (200), producedby a packaging cell (100) and a resting T cell (300) transfected by thereplication incompetent recombinant retroviral particle (200). Theelements on the surface of the replication incompetent recombinantretroviral particle (200), bind to receptors and/or ligands on thesurface of a resting T cell. The pseudotyping element can include, innon-limiting embodiments, a binding polypeptide and a fusogenicpolypeptide (in non-limiting embodiments, a Measles Virus hemagglutinin(H) polypeptide and a Measles Virus fusion (F) polypeptide, orcytoplasmic domain deletion variants thereof) that facilitate thebinding and fusion of the replication incompetent recombinant retroviralparticle (200), to the T cell. In non-limiting embodiments, thereplication incompetent recombinant retroviral particle (200), includeson its surface an activation element (in non-limiting embodiments anactivation element that has a polypeptide capable of binding to CD28 anda polypeptide capable of binding to CD3) that is capable of activatingthe resting T cell by engaging the T-cell receptor complex andoptionally a co-receptor (320). Furthermore, membrane-bound cytokines(in non-limiting embodiments, an IL-7 DAF fusion polypeptide) present onthe surface of the replication incompetent recombinant retroviralparticle (200), bind to IL-7Rα (310) on the surface of the resting Tcell. The replication incompetent recombinant retroviral particle (200),fuses with the T cell, and polynucleotides that encode the firstengineered signaling polypeptide that includes the lymphoproliferativeelement (in illustrative embodiments, a constitutively active IL-7Ra)(370), are reverse transcribed in the cytosol prior to migrating to thenucleus to be incorporated into the DNA of the activated T cell. Not tobe limited by theory, in some non-limiting embodiments, Src-FLAG-Vpx(250) packaged with the virus enters the cytosol of the resting T cellsand promotes the degradation of SAMHD1 (350), resulting in an increasedpool of cytoplasmic dNTPs available for reverse transcription. In someembodiments, the polynucleotides can also encode a second engineeredsignaling polypeptide that includes a CAR (360). In some embodiments,the lymphoproliferative element is expressed when a compound binds to acontrol element that regulates its expression (in non-limiting example,the control element is a riboswitch that binds a nucleoside analog). Insome embodiments, expression of the CAR is also regulated by the controlelement. Part (330) is SLAM and CD46. Part (340) is CD3.

FIGS. 3A-3E show schematics of non-limiting, exemplary vector constructsfor transfecting packaging cells to produce replication incompetentrecombinant retroviral particles described herein. FIG. 3A shows aconstruct containing a polynucleotide sequence encoding an FRB domainfused to the NFκB p65 activator domain (p65 AD) and ZFHD1 DNA bindingdomain fused to three FKBP repeats that is constitutively expressed. Theconstruct in FIG. 3A also includes HIV1 REV and Vpx as a SrcFlagVpxfusion under the rapamycin-inducible ZFHD1/p65 AD promoter. FIG. 3Bshows a construct containing a polynucleotide encoding an rtTA sequenceunder the control of the ZFHD1/p65 AD promoter. FIG. 3C shows aconstruct containing a polynucleotide encoding a puromycin resistancegene flanked by loxP sites and the extracellular MYC tag flanked bylox2272 sites. Both selectable markers are under the control of a BiTREpromoter, which is flanked by FRT sites. FIG. 3D shows a construct thatcontains a polynucleotide encoding RFP flanked by loxP sites that isunder the control of a TRE promoter and a single FRT site between theTRE promoter and the 5′ loxP site of RFP. FIG. 3E shows a constructcontaining a polynucleotide encoding GFP flanked by loxP sites that isunder the control of the TRE promoter and a single FRT site between theTRE promoter and the 5′ loxP site of GFP. The constructs in FIGS. 3C-3Efunction as landing pads for other polynucleotide sequences to insertinto the genome of the packaging cell line.

FIGS. 4A-4C show schematics of non-limiting, exemplary vector constructsfor transfecting packaging cells to produce replication incompetentrecombinant retroviral particles described herein. FIG. 4A shows aconstruct containing a tricistronic polynucleotide encoding anti-CD3(clone UCHT1) scFvFc with a CD14 GPI anchor attachment site, CD80 extracellular domain (ECD) capable of binding CD28 with a CD16B GPI anchorattachment site, and IL-7 fused to decay-accelerating factor (DAF) withtransposon sequences flanking the polynucleotide region for integrationinto the HEK293S genome. FIG. 4B shows a construct containing apolynucleotide with a BiTRE promoter and a polynucleotide regionencoding the gag and pol polypeptides in one direction and apolynucleotide region encoding the measles virus FΔx and HΔy proteins inthe other direction. FIG. 4C shows a construct containing apolynucleotide sequence encoding a CAR and the lymphoproliferativeelement IL7Rα-insPPCL under the control of a CD3Z promoter which is notactive in HEK293S cells, wherein the CAR and IL7Rα-insPPCL are separatedby a polynucleotide sequence encoding a T2A ribosomal skip sequence andthe IL7Rα-insPPCL has an acyclovir riboswitch controlled ribozyme. TheCAR-containing construct further includes cPPT/CTS, an RRE sequence, anda polynucleotide sequence encoding HIV-1 Psi (Ψ). The entirepolynucleotide sequence on the CAR-containing construct to be integratedinto the genome is flanked by FRT sites.

FIGS. 5A-5C show molecular structures of acyclovir (FIG. 5A),penciclovir (FIG. 5B), and 2′-deoxyguanonsine (FIG. 5C) asrepresentative nucleoside analogues for selective riboswitch control.

FIG. 6 represents the Mesoplasma florum type I-A deoxyguanosineriboswitch regulatory region and associated gene product. The sequenceis the reverse complement of M. florum L1 genomic DNA (AE017263.1)nt624396 to nt625670 which is same as M. florum W37 genomic DNA(CP006778.1) nt636277 to nt 637550. The deoxyguanosine binding aptamersequence used for initial screen indicated in bold and underline. Thedownstream gene product (Ribonucleotide reductase of class Ib (aerobic),beta subunit) is indicated in capital letters.

FIG. 7 represents the M. florum type I-A deoxyguanosine riboswitchaptamer regions targeted for directed evolution strategy. Nucleotideswithin empty ovals were targeted for randomization. Nucleotides withinstriped ovals were targeted for insertion/deletion and randomization.

FIGS. 8A and 8B represent the M. florum type I-A deoxyguanosineriboswitch aptamer screening library. In FIG. 8A, nucleotides withinboxes with solid lines are sequence regions targeted for randomizationand nucleotides within boxes with dashed lines are sequence regionstargeted for insertion/deletion and randomization. FIG. 8B showspossible sequences generated through mutation (“random nucleotides(“N”)) and deletion/insertion.

FIG. 9 represents the M. florum type I-A deoxyguanosine riboswitchaptamer oligo library synthesized as a reverse complement withadditional base pairs added to allow for PCR amplification and T7promoter addition for in vitro transcription for library screening. Thecorresponding T7 promoter amplification primer and reverse amplificationprimer are also shown.

FIG. 10 represents the Bacillus subtilis guanosine xpt riboswitchregulatory region and associated gene product. The sequence is thereverse complement of B. subtilis subsp. subtilis 6051-HGW genomic DNA(CP003329.1) nt2319439 to nt2320353. The guanosine binding aptamersequence used for initial screen indicated in bold and underline. Thedownstream gene product (Xanthine phosphoribosyltransferase xpt) isindicated in capital letters.

FIG. 11 represents the B. subtilis guanosine xpt riboswitch aptamerregions targeted for directed evolution strategy. Nucleotides withinempty ovals were targeted for randomization. Nucleotides within stripedovals were targeted for insertion/deletion and randomization

FIGS. 12A and 12B represent the B. subtilis guanosine xpt riboswitchaptamer screening library. In FIG. 12A, nucleotides within boxes withsolid lines are sequence regions targeted for randomization andnucleotides within boxes with dashed lines are sequence regions targetedfor insertion/deletion and randomization. FIG. 12B shows possiblesequences generated through mutation (random nucleotides (“N”)) anddeletion/insertion.

FIG. 13 represents the B. subtilis guanosine xpt riboswitch aptameroligo library synthesized as a reverse complement with additional basepairs added to allow for PCR amplification and T7 promoter addition forin vitro transcription for library screening. The corresponding T7promoter amplification primer and reverse amplification primer are alsoshown.

FIG. 14 shows the selection library construction. The library wasconstructed on the basis of known guanosine- and deoxyguanosine-bindingRNA (Pikovskaya, 2013).

FIG. 15 shows an illustration of graphene oxide (GrO) aptamer selection.In step (1), RNA was transcribed and purified. In step (2), purified RNAwas eluted. In step (3), aptamers were incubated with counter-targetsand buffer. In step (4), sequences bound to counter-targets or buffercomponents were removed with graphene oxide. In step (5), centrifugationpartitioned the non-specifically-responsive species within thesupernatant, which is then discarded. Two additional 5-minute washesremoved most of the residual counter-target-binding and buffer-bindingsequences. In step (6), a solution of acyclovir in 1×selection bufferwas added to the GrO-bound library for positive selection so potentialaptamer sequences desorb from the GrO through interaction with thepositive target. In step (7), a final centrifugation step separates thetarget-binding sequences in the supernatant from the non-responsivesequences still adsorbed to the GrO. In step (8) selected sequences werereverse-transcribed, then the library was amplified through PCR, thentranscribed to generate library for the next selection round.

FIG. 16 shows an illustration of graphene oxide parallel assessment.Enriched libraries undergoing parallel assessment were divided into fourequal portions. Library samples were then added to graphene oxide andallowed to incubate to load the library on the graphene oxide. Two5-minute washes were used to remove non-binding material. For thepositive (acyclovir) and special target (penciclovir) sample, eachtarget was prepared separately in 1×selection buffer to 1 μM; thecounter target replaced the positive target with 10 μM of eachcounter-target in solution; the negative sample replaced the positivetarget with an equal volume of nuclease-free water. Samples were thencombined with their respective graphene oxide preparations andincubated. Post-incubation, samples were centrifuged to recover theirsupernatants, and library recovery was determined by NanoDrop-1000spectrophotometer reading (Thermo Fisher Scientific; Wilmington, DE).Remaining library sample was analyzed on denaturing PAGE. Images of thegels were taken after staining/destaining with Gel-Star. Bandscorresponding to expected library size were recovered for a follow-upround of parallel assessment, with positive target acyclovir replacingcounter-targets for the negative, counter, and special target samples'pre-loading incubation. Material recovered from the second parallelassessment was used for sequencing and analysis.

FIG. 17 shows seven aptamer candidates against acyclovir. The freeenergy for each aptamer was computed at 37° C. and 1 M Na+ by Quikfold3.0 (Zuker 2003). Sequences were identified using proprietaryalgorithms. The underlined regions in each sequence are the PCR primerannealing regions.

FIG. 18 shows seven aptamer candidates against penciclovir. The freeenergy for each aptamer was computed at 37° C. and 1 M Na+ by Quikfold3.0 (Zuker 2003). Sequences were identified using proprietaryalgorithms. The underlined regions in each sequence are the PCR primerannealing regions.

FIG. 19A provides a schematic of IL7Rα variants tested forlymphoproliferative/survival activity when expressed in PBMCs. FIG. 19Bprovides a bar graph showing percent viability of PBMCs in the presenceand absence of IL-2.

FIG. 20 shows a schematic of the lentiviral expression vector encodingGFP, an anti-CD19 chimeric antigen receptor, and an eTAG referred toherein as F1-0-03.

FIG. 21A and FIG. 21B show a histogram of the percentage (%) CD3+GFP+cells in the total CD3+ population and a histogram of the absolute cellcount per well of the CD3+GFP+ population, respectively, at 3, 6, 9, 13and 17 days after transduction of freshly isolated and unstimulatedPBMCs from Donor 12M, for 14 h with the indicated lentiviral particles.Each bar represents the mean+/−SD of duplicates.

FIG. 22A and FIG. 22B show a histogram of (%) CD3+GFP+ cells in thetotal CD3+ population and a histogram of the absolute cell count perwell of the CD3+GFP+ population, respectively, at 3 and 6 days aftertransduction of freshly isolated and unstimulated PBMCs from Donor 13F,for 14 h, with the indicated lentiviral particles. Please note that “A”shows results using VSV-G pseudotyped lentiviral particles (triplicateexperiments); “B” shows results using VSV-G pseudotyped lentiviralparticles with OKT3 Ab (1 μg/mL) added to the transduction medium(duplicate experiments); “C” shows results using VSV-G pseudotypedlentiviral particles expressing GPI-anchored UCHT1scFvFc on theirsurface (triplicate experiments); and “D” shows results using VSV-Gpseudotyped lentiviral particles expressing GPI anchored UCHT1scFvFc andGPI-anchored CD80, or a functional extracellular fragment thereof, ontheir surface (duplicate experiments). Each bar represents the mean+/−SDof duplicates or triplicates, as indicated in FIG. 22A.

FIG. 23A and FIG. 23B show a histogram of percentage (%) CD3+GFP+ cellsin the total CD3+ population and a histogram of the absolute cell countper well of the CD3+GFP+ population, respectively, at 3, 6 and 9 daysafter transduction of freshly isolated and unstimulated PBMCs from Donor12M for the indicated time of exposure (2-20 h), with the indicatedlentiviral particles. Transduction was performed in a plate or a shakerflask as indicated. Each bar represents the mean+/−SD of duplicates forlentiviral particles pseudotyped with VSV-G (“[VSV-G]”); the otherexperiments did not have replicates.

FIG. 24A is a schematic of the lentiviral vector backbone F1-0-02including a transgene expression cassette driving expression of GFP andeTag and a synthetic EF-1alpha promoter and intron A upstream of theGFP. FIG. 24B shows insertion of the miRNAs into EF1alpha intron A ofthe F1-0-02 backbone. “1” represents the EF1alpha overlap; “2”represents a 5′ arm; “3” represents the miRNA1 5′ stem; “4” represents aloop; “5” represents the miRNA1 3′ stem; “6” represents a 3′ arm; “7”represents a linker; “8” represents the miRNA2 5′ stem; “9” representsthe miRNA2 3′ stem; “10” represents the miRNA3 5′ stem; “1 1” representsthe miRNA3 3′ stem; “12” represents the miRNA4 5′ stem; and “13”represents the miRNA4 3′ stem.

FIG. 25 is a graph showing that the miRNAs targeting CD3zeta that are inthe EF-1alpha promoter intron are able to knockdown expression of theCD3 complex.

FIG. 26 is a histogram showing the ΔΔCt of samples transduced withmiR-TCRα containing replication incompetent lentiviral particles. TheΔΔCt values are representative of the amount of processed miR-TCRα miRNAin each transduced sample relative to the non-transduced control.

FIGS. 27A-C are graphs showing the percent specific lysis of CHO-Target1 cells with and without treatment with a pH-modulating pharmacologicagent. In FIG. 27A, the CHO-Target 1 cells were initially at pH 6.7 andexperimental wells (solid line) and control cells (dashed line) weretreated with or without NaHCO₃, respectively, at the time indicated bythe arrow. In FIG. 27B, the CHO-Target 1 cells were initially at pH 6.7and experimental wells (solid line) and control cells (dashed line) weretreated with or without NaOH, respectively, at the time indicated by thearrow. In FIG. 27C, the CHO-Target 1 cells were initially at pH 7.4 andexperimental wells (solid line) and control cells (dashed line) weretreated with or without HCl, respectively

FIG. 28 is a graph showing the heat flux versus time for F1A-795 in theabsence (circles) or presence (squares) of acyclovir as measured by DSC.

FIG. 29 is a graph showing the RFU percentage from ProSense FAST probein CHO-xenograft tumor bearing mice before and after administration ofPBS or bicarbonate.

FIG. 30 is a schematic of a non-limiting, exemplary transgene expressioncassette containing a polynucleotide sequence encoding a CAR and acandidate chimeric lymphoproliferative element (CLE) of Libraries 1A,1.1A, and 1.1B.

FIG. 31 is a schematic of a non-limiting, exemplary transgene expressioncassette containing a polynucleotide sequence encoding a candidate CLEof Libraries 2B and 2.1B.

FIG. 32 is a schematic of a non-limiting, exemplary transgene expressioncassette containing a polynucleotide sequence encoding a CAR and acandidate CLE of Libraries 3A, 3B, 3.1A, and 3.1B.

FIG. 33 is a schematic of a non-limiting, exemplary transgene expressioncassette containing a polynucleotide sequence a candidate CLE ofLibraries 4B and 4.1 B.

FIG. 34A shows a histogram of the percentage (%) CD3+GFP+ cells in theLive CD3+ population, and FIG. 34B shows a histogram of the absolutecell count per uL of the total live population, at day 3post-transduction of freshly isolated and unstimulated PBMCs from Donor18, with the indicated lentiviral particles. Each bar represents themean+/−SD of duplicates.

FIG. 35 is a graph showing the fold expansion of PBMCs transduced withlentiviral particles encoding individual CLEs and cultured for 35 daysin the absence of exogenous cytokines.

FIG. 36 is a graph showing the fold expansion of PBMCs transduced withlentiviral particles encoding an anti-CD19 CAR construct and individualCLEs and cultured for 35 days in the presence of donor matched PBMCs butin the absence of exogenous cytokines.

FIG. 37 is a graph showing the efficiency by which the indicatedlentiviral particle transduced resting PBMCs in 4 hours. Transductionefficiency was measured as the % CAR+ PBMCs after 6 days in culture inthe absence of exogenous cytokines as determined by FACS. Eachlentiviral particle encoded a CAR and a CLE. Lentiviral particlestransduced with F1-1-228U and F1-3-219U displayed UCHT1scFvFc-GPI ontheir surface.

FIG. 38A and FIG. 38B are graphs showing a time course of the totalnumber of viable cells after resting PBMCs were transduced with theindicated lentiviral particle for 4 hours and cultured in vitro in theabsence of exogenous cytokines for 6 days. Each lentiviral particleencoded a CAR and a CLE. Lentiviral particles transduced with F1-1-228Uand F1-3-219U displayed UCHT1scFvFc-GPI on their surface.

FIGS. 39A, 39B, and 39C are graphs showing a time course of the copiesof lentiviral genome per pg of genomic DNA from the blood oftumor-bearing NSG mice dosed with human PBMCs transduced with theindicated lentiviral particle for 4 hours and injected intravenouslywithout the PBMCs having been expanded ex vivo. Each lentiviral particleencoded a CAR. F1-1-228, F1-1-228U, F1-3-219, and F1-3-219U also encodeda CLE. Lentiviral particles transduced with F1-1-228U and F1-3-219Udisplayed UCHT1scFvFc-GPI on their surface.

FIG. 40 is a graph showing the number of CAR+ cells per 200 μl of bloodof tumor-bearing NSG mice dosed with human PBMCs transduced with theindicated lentiviral particle for 4 hours and injected intravenouslywithout the PBMCs having been expanded ex vivo. Blood was sampled at thetime the mice were euthanized. Each lentiviral particle encoded a CAR.F1-1-228, F1-1-228U, F1-3-219, and F1-3-219U also encoded a CLE.Lentiviral particles transduced with F1-1-228U and F1-3-219U displayedUCHT1scFvFc-GPI on their surface.

FIG. 41A is a graph showing the mean tumor volume of CHO-ROR2 tumors inNSG mice dosed intravenously with PBS or human PBMCs transduced with theindicated lentiviral particle encoding an anti-ROR2 MRB CAR and a CLEfor 4 hours without the PBMCs having been expanded ex vivo. 41B is agraph showing the mean tumor volume of Raji tumors in NSG mice dosedintravenously with PBS or human PBMCs transduced with the indicatedlentiviral particle encoding an anti-CD19 CAR and a CLE for 4 hourswithout the PBMCs having been expanded ex vivo. Lentiviral particlestransduced with F1-1-228U and F1-3-219U displayed UCHT1scFvFc-GPI ontheir surface.

FIG. 42A is a non-limiting diagram of a RIR retroviral particle with anactivation element on the surface of the retroviral particle.

FIG. 42B is a schematic of a non-limiting, exemplary transgeneexpression cassette containing a CAR and a lymphoproliferative element.

FIG. 43 is a graph showing the number of transduced cells (CD3+/FLAG+)per μl at Day 6 after transduction of unstimulated PBMCS by RIRretroviral particles A, B, C, or D for various times, with or withoutreverse transcriptase or integrase inhibitors.

FIG. 44 is a graph showing the number of transduced PBMCs as measured byFACS for CAR expression (by FLAG tag) after 7, 14, and 21 days ofculture in vitro in the absence of exogenous cytokines.

FIG. 45A is a graph showing the lentiviral copies per pg of genomic DNAisolated from blood of the MRB-ROR2 CAR-expressing PBMC dosed mice, byqPCR as a readout for the number of transduced cells that weremanufactured using the rapid point-of-care (rPOC) process with RIRretroviral particles E and F, as indicated, without ex vivo cellexpansion.

FIG. 45B is a graph showing the lentiviral copies per pg of genomic DNAisolated from blood of the MRB-ROR2 CAR-expressing PBMC dosed mice, byqPCR as a readout for the number of transduced cells that weremanufactured using a traditional protocol of activation, transduction,and ex vivo cell manufacturing process with retroviral particles G.

DEFINITIONS

As used herein, the term “chimeric antigen receptor” or “CAR” or “CARs”refers to engineered receptors, which graft an antigen specificity ontocells, for example T cells, NK cells, macrophages, and stem cells. TheCARs of the invention include at least one antigen-specific targetingregion (ASTR), a transmembrane Domain (TM), and an intracellularactivating domain (IAD) and can include a stalk, and one or moreco-stimulatory domains (CSDs). In another embodiment, the CAR is abispecific CAR, which is specific to two different antigens or epitopes.After the ASTR binds specifically to a target antigen, the IAD activatesintracellular signaling. For example, the IAD can redirect T cellspecificity and reactivity toward a selected target in anon-MHC-restricted manner, exploiting the antigen-binding properties ofantibodies. The non-MHC-restricted antigen recognition gives T cellsexpressing the CAR the ability to recognize an antigen independent ofantigen processing, thus bypassing a major mechanism of tumor escape.Moreover, when expressed in T cells, CARs advantageously do not dimerizewith endogenous T cell receptor (TCR) alpha and beta chains.

As used herein, the term “microenvironment” means any portion or regionof a tissue or body that has constant or temporal, physical, or chemicaldifferences from other regions of the tissue or regions of the body. Forexample, a “tumor microenvironment” as used herein refers to theenvironment in which a tumor exists, which is the non-cellular areawithin the tumor and the area directly outside the tumorous tissue butdoes not pertain to the intracellular compartment of the cancer cellitself. The tumor microenvironment can refer to any and all conditionsof the tumor milieu including conditions that create a structural and orfunctional environment for the malignant process to survive and/orexpand and/or spread. For example, the tumor microenvironment caninclude alterations in conditions such as, but not limited to, pressure,temperature, pH, ionic strength, osmotic pressure, osmolality, oxidativestress, concentration of one or more solutes, concentration ofelectrolytes, concentration of glucose, concentration of hyaluronan,concentration of lactic acid or lactate, concentration of albumin,levels of adenosine, levels of R-2-hydroxyglutarate, concentration ofpyruvate, concentration of oxygen, and/or presence of oxidants,reductants, or co-factors, as well as other conditions a skilled artisanwill understand.

As used interchangeably herein, the terms “polynucleotide” and “nucleicacid” refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. Thus, this term includes, butis not limited to, single-, double-, or multi-stranded DNA or RNA,genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine andpyrimidine bases or other natural, chemically or biochemically modified,non-natural, or derivatized nucleotide bases.

As used herein, the term “antibody” includes polyclonal and monoclonalantibodies, including intact antibodies and fragments of antibodieswhich retain specific binding to antigen. The antibody fragments can be,but are not limited to, fragment antigen binding (Fab) fragments, Fab′fragments, F(ab′)₂ fragments, Fv fragments, Fab′-SH fragments, (Fab′)₂Fv fragments, Fd fragments, recombinant IgG (rIgG) fragments,single-chain antibody fragments, including single-chain variablefragments (scFv), divalent scFv's, trivalent scFv's, and single domainantibody fragments (e.g., sdAb, sdFv, nanobody). The term includesgenetically engineered and/or otherwise modified forms ofimmunoglobulins, such as intrabodies, peptibodies, chimeric antibodies,single-chain antibodies, fully human antibodies, humanized antibodies,fusion proteins including an antigen-specific targeting region of anantibody and a non-antibody protein, heteroconjugate antibodies,multispecific, e.g., bispecific, antibodies, diabodies, triabodies, andtetrabodies, tandem di-scFv's, and tandem tri-scFv's. Unless otherwisestated, the term “antibody” should be understood to include functionalantibody fragments thereof. The term also includes intact or full-lengthantibodies, including antibodies of any class or sub-class, includingIgG and sub-classes thereof, IgM, IgE, IgA, and IgD.

As used herein, the term “antibody fragment” includes a portion of anintact antibody, for example, the antigen binding or variable region ofan intact antibody. Examples of antibody fragments include Fab, Fab′,F(ab′)₂, and Fv fragments; diabodies; linear antibodies (Zapata et al.,Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules;and multispecific antibodies formed from antibody fragments. Papaindigestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fe” fragment, a designation reflecting the abilityto crystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen combining sites and is still capable of cross-linkingantigen.

As used interchangeably herein, the terms “single-chain Fv,” “scFv,” or“sFv” antibody fragments include the V_(H) and V_(L) domains ofantibody, wherein these domains are present in a single polypeptidechain. In some embodiments, the Fv polypeptide further includes apolypeptide linker or spacer between the V_(H) and V_(L) domains, whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

As used herein, “naturally occurring” VH and VL domains refer to VH andVL domains that have been isolated from a host without further molecularevolution to change their affinities when generated in an scFv formatunder specific conditions such as those disclosed in U.S. Pat. No.8,709,755 B2 and application WO/2016/033331A1.

As used herein, the term “affinity” refers to the equilibrium constantfor the reversible binding of two agents and is expressed as adissociation constant (Kd). Affinity can be at least I-fold greater, atleast 2-fold greater, at least 3-fold greater, at least 4-fold greater,at least 5-fold greater, at least 6-fold greater, at least 7-foldgreater, at least 8-fold greater, at least 9-fold greater, at least10-fold greater, at least 20-fold greater, at least 30-fold greater, atleast 40-fold greater, at least 50-fold greater, at least 60-foldgreater, at least 70-fold greater, at least 80-fold greater, at least90-fold greater, at least 100-fold greater, or at least 1000-foldgreater, or more, than the affinity of an antibody for unrelated aminoacid sequences. Affinity of an antibody to a target protein can be, forexample, from about 100 nanomolar (nM) to about 0.1 nM, from about 100nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar(fM) or more. As used herein, the term “avidity” refers to theresistance of a complex of two or more agents to dissociation afterdilution. The terms “immunoreactive” and “preferentially binds” are usedinterchangeably herein with respect to antibodies and/or antigen-bindingfragments.

As used herein, the term “binding” refers to a direct associationbetween two molecules, due to, for example, covalent, electrostatic,hydrophobic, and ionic and/or hydrogen-bond interactions, includinginteractions such as salt bridges and water bridges. Non-specificbinding would refer to binding with an affinity of less than about 10-7M, e.g., binding with an affinity of 10⁻⁶ M, 10-5 M, 10-4 M, etc.

As used herein, reference to a “cell surface expression system” or “cellsurface display system” refers to the display or expression of a proteinor portion thereof on the surface of a cell. Typically, a cell isgenerated that expresses proteins of interest fused to a cell-surfaceprotein. For example, a protein is expressed as a fusion protein with atransmembrane domain.

As used herein, the term “element” includes polypeptides, includingfusions of polypeptides, regions of polypeptides, and functional mutantsor fragments thereof and polynucleotides, including microRNAs andshRNAs, and functional mutants or fragments thereof.

As used herein, the term “region” is any segment of a polypeptide orpolynucleotide.

As used herein, a “domain” is a region of a polypeptide orpolynucleotide with a functional and/or structural property.

As used herein, the terms “stalk” or “stalk domain” refer to a flexiblepolypeptide connector region providing structural flexibility andspacing to flanking polypeptide regions and can consist of natural orsynthetic polypeptides. A stalk can be derived from a hinge or hingeregion of an immunoglobulin (e.g., IgG1) that is generally defined asstretching from Glu216 to Pro230 of human IgG1 (Burton (1985) Molec.Immunol., 22:161-206). Hinge regions of other IgG isotypes may bealigned with the IgG1 sequence by placing the first and last cysteineresidues forming inter-heavy chain disulfide (S—S) bonds in the samepositions. The stalk may be of natural occurrence or non-naturaloccurrence, including but not limited to an altered hinge region, asdisclosed in U.S. Pat. No. 5,677,425. The stalk can include a completehinge region derived from an antibody of any class or subclass. Thestalk can also include regions derived from CD8, CD28, or otherreceptors that provide a similar function in providing flexibility andspacing to flanking regions.

As used herein, the term “isolated” means that the material is removedfrom its original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally-occurring polynucleotideor polypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

As used herein, a “polypeptide” is a single chain of amino acid residueslinked by peptide bonds. A polypeptide does not fold into a fixedstructure nor does it have any posttranslational modification. A“protein” is a polypeptide that folds into a fixed structure.“Polypeptides” and “proteins” are used interchangeably herein.

As used herein, a polypeptide may be “purified” to remove contaminantcomponents of a polypeptide's natural environment, e.g. materials thatwould interfere with diagnostic or therapeutic uses for the polypeptidesuch as, for example, enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. A polypeptide can be purified (1) to greaterthan 90%, greater than 95%, or greater than 98%, by weight of antibodyas determined by the Lowry method, for example, more than 99% by weight,(2) to a degree sufficient to obtain at least 15 residues of N-terminalor internal amino acid sequence by use of a spinning cup sequenator, or(3) to homogeneity by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) under reducing or nonreducing conditionsusing Coomassie blue or silver stain.

As used herein, the term “immune cells” generally includes white bloodcells (leukocytes) which are derived from hematopoietic stem cells (HSC)produced in the bone marrow. “Immune cells” includes, e.g., lymphocytes(T cells, B cells, natural killer (NK) cells) and myeloid-derived cells(neutrophil, eosinophil, basophil, monocyte, macrophage, dendriticcells).

As used herein, “T cell” includes all types of immune cells expressingCD3 including T-helper cells (CD4⁺ cells), cytotoxic T cells (CD8⁺cells), T-regulatory cells (Treg) and gamma-delta T cells.

As used herein, a “cytotoxic cell” includes CD8⁺ T cells, natural-killer(NK) cells, NK-T cells, γδ T cells, a subpopulation of CD4⁺ cells, andneutrophils, which are cells capable of mediating cytotoxicityresponses.

As used herein, the term “stem cell” generally includes pluripotent ormultipotent stem cells. “Stem cells” includes, e.g., embryonic stemcells (ES); mesenchymal stem cells (MSC); induced-pluripotent stem cells(iPS); and committed progenitor cells (hematopoietic stem cells (HSC);bone marrow derived cells, etc.).

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease in a mammal, e.g., in a human, and includes: (a)preventing the disease from occurring in a subject which may bepredisposed to the disease but has not yet been diagnosed as having it;(b) inhibiting the disease, i.e., arresting its development; and (c)relieving the disease, i.e., causing regression of the disease.

As used interchangeably herein, the terms “individual”, “subject”,“host”, and “patient” refer to a mammal, including, but not limited to,humans, murines (e.g., rats, mice), lagomorphs (e.g., rabbits),non-human primates, humans, canines, felines, ungulates (e.g., equines,bovines, ovines, porcines, caprines), etc.

As used herein, the terms “therapeutically effective amount” or“efficacious amount” refers to the amount of an agent, or combinedamounts of two agents, that, when administered to a mammal or othersubject for treating a disease, is sufficient to affect such treatmentfor the disease. The “therapeutically effective amount” will varydepending on the agent(s), the disease and its severity and the age,weight, etc., of the subject to be treated.

As used herein, the term “evolution” or “evolving” refers to using oneor more methods of mutagenesis to generate a different polynucleotideencoding a different polypeptide, which is itself an improved biologicalmolecule and/or contributes to the generation of another improvedbiological molecule. “Physiological” or “normal” or “normalphysiological” conditions are conditions such as, but not limited to,pressure, temperature, pH, ionic strength, osmotic pressure, osmolality,oxidative stress, concentration of one or more solutes, concentration ofelectrolytes, concentration of glucose, concentration of hyaluronan,concentration of lactic acid or lactate, concentration of albumin,levels of adenosine, levels of R-2-hydroxyglutarate, concentration ofpyruvate, concentration of oxygen, and/or presence of oxidants,reductants, or co-factors, as well as other conditions, that would beconsidered within a normal range at the site of administration, or atthe tissue or organ at the site of action, to a subject.

As used herein, a “genetically modified cell” includes cells thatcontain exogenous nucleic acids whether or not the exogenous nucleicacids are integrated into the genome of the cell.

A “polypeptide” as used herein can include part of or an entire proteinmolecule as well as any posttranslational or other modifications.

A pseudotyping element as used herein can include a “bindingpolypeptide” that includes one or more polypeptides, typicallyglycoproteins, that identify and bind the target host cell, and one ormore “fusogenic polypeptides” that mediate fusion of the retroviral andtarget host cell membranes, thereby allowing a retroviral genome toenter the target host cell. The “binding polypeptide” as used herein,can also be referred to as a “T cell and/or NK cell binding polypeptide”or a “target engagement element,” and the “fusogenic polypeptide” canalso be referred to as a “fusogenic element”.

A “resting” lymphocyte, such as for example, a resting T cell, is alymphocyte in the GO stage of the cell cycle that does not expressactivation markers such as Ki-67. Resting lymphocytes can include naïveT cells that have never encountered specific antigen and memory T cellsthat have been altered by a previous encounter with an antigen. A“resting” lymphocyte can also be referred to as a “quiescent”lymphocyte.

As used herein, “lymphodepletion” involves methods that reduce thenumber of lymphocytes in a subject, for example by administration of alymphodepletion agent. Lymphodepletion can also be attained by partialbody or whole body fractioned radiation therapy. A lymphodepletion agentcan be a chemical compound or composition capable of decreasing thenumber of functional lymphocytes in a mammal when administered to themammal. One example of such an agent is one or more chemotherapeuticagents. Such agents and dosages are known, and can be selected by atreating physician depending on the subject to be treated. Examples oflymphodepletion agents include, but are not limited to, fludarabine,cyclophosphamide, cladribine, denileukin diftitox, or combinationsthereof.

RNA interference (RNAi) is a biological process in which RNA moleculesinhibit gene expression or translation by neutralizing targeted RNAmolecules. The RNA target may be mRNA, or it may be any other RNAsusceptible to functional inhibition by RNAi. As used herein, an“inhibitory RNA molecule” refers to an RNA molecule whose presencewithin a cell results in RNAi and leads to reduced expression of atranscript to which the inhibitory RNA molecule is targeted. Aninhibitory RNA molecule as used herein has a 5′ stem and a 3′ stem thatis capable of forming an RNA duplex. The inhibitory RNA molecule can be,for example, a miRNA (either endogenous or artificial) or a shRNA, aprecursor of a miRNA (i.e. a Pri-miRNA or Pre-miRNA) or shRNA, or adsRNA that is either transcribed or introduced directly as an isolatednucleic acid, to a cell or subject.

As used herein, “double stranded RNA” or “dsRNA” or “RNA duplex” refersto RNA molecules that are comprised of two strands. Double-strandedmolecules include those comprised of two RNA strands that hybridize toform the duplex RNA structure or a single RNA strand that doubles backon itself to form a duplex structure. Most, but not necessarily all ofthe bases in the duplex regions are base-paired. The duplex regioncomprises a sequence complementary to a target RNA. The sequencecomplementary to a target RNA is an antisense sequence, and isfrequently from 18 to 29, from 19 to 29, from 19 to 21, or from 25 to 28nucleotides long, or in some embodiments between 18, 19, 20, 21, 22, 23,24, 25 on the low end and 21, 22, 23, 24, 25, 26, 27, 28 29, or 30 onthe high end, where a given range always has a low end lower than a highend. Such structures typically include a 5′ stem, a loop, and a 3′ stemconnected by a loop which is contiguous with each stem and which is notpart of the duplex. The loop comprises, in certain embodiments, at least3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In other embodiments the loopcomprises from 2 to 40, from 3 to 40, from 3 to 21, or from 19 to 21nucleotides, or in some embodiments between 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 on the low end and 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40 on the high end, wherea given range always has a low end lower than a high end.

The term “microRNA flanking sequence” as used herein refers tonucleotide sequences including microRNA processing elements. MicroRNAprocessing elements are the minimal nucleic acid sequences whichcontribute to the production of mature microRNA from precursor microRNA.Often these elements are located within a 40 nucleotide sequence thatflanks a microRNA stem-loop structure. In some instances the microRNAprocessing elements are found within a stretch of nucleotide sequencesof between 5 and 4,000 nucleotides in length that flank a microRNAstem-loop structure.

The term “linker” when used in reference to a multiplex inhibitory RNAmolecule refers to a connecting means that joins two inhibitory RNAmolecules.

As used herein, a “recombinant retrovirus” refers to a non-replicable,or “replication incompetent”, retrovirus unless it is explicitly notedas a replicable retrovirus. The terms “recombinant retrovirus” and“recombinant retroviral particle” are used interchangeably herein. Suchretrovirus/retroviral particle can be any type of retroviral particleincluding, for example, gamma retrovirus, and in illustrativeembodiments, lentivirus. As is known, such retroviral particles, forexample lentiviral particles, typically are formed in packaging cells bytransfecting the packing cells with plasmids that include packagingcomponents such as Gag, Pol and Rev, an envelope or pseudotyping plasmidthat encodes a pseudotyping element, and a transfer, genomic, orretroviral (e.g. lentiviral) expression vector, which is typically aplasmid on which a gene(s) or other coding sequence of interest isencoded. Accordingly, a retroviral (e.g. lentiviral) expression vectorincludes sequences (e.g. a 5′ LTR and a 3′ LTR flanking e.g. a psipackaging element and a target heterologous coding sequence) thatpromote expression and packaging after transfection into a cell. Theterms “lentivirus” and “lentiviral particle” are used interchangeablyherein.

A “framework” of a miRNA consists of “5′ microRNA flanking sequence”and/or “3′ microRNA flanking sequence” surrounding a miRNA and, in somecases, a loop sequence that separates the stems of a stem-loop structurein a miRNA. In some examples, the “framework” is derived from naturallyoccurring miRNAs, such as, for example, miR-155. The terms “5′ microRNAflanking sequence” and “5′ arm” are used interchangeably herein. Theterms “3′ microRNA flanking sequence” and “3′ arm” are usedinterchangeably herein.

As used herein, the term “miRNA precursor” refers to an RNA molecule ofany length which can be enzymatically processed into an miRNA, such as aprimary RNA transcript, a pri-miRNA, or a pre-miRNA.

It is to be understood that the present disclosure and the aspects andembodiments provided herein, are not limited to particular examplesdisclosed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of disclosingparticular examples and embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention. Whenmultiple low and multiple high values for ranges are given that overlap,a skilled artisan will recognize that a selected range will include alow value that is less than the high value. All headings in thisspecification are for the convenience of the reader and are notlimiting.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “achimeric antigen receptor” includes a plurality of such chimeric antigenreceptors and equivalents thereof known to those skilled in the art, andso forth. It is further noted that the claims may be drafted to excludeany optional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

DETAILED DESCRIPTION

The present disclosure overcomes prior art challenges by providingimproved methods and compositions for genetically modifying lymphocytes,for example NK cells and in illustrative embodiments, T cells. Forexample, some of these methods do not include prior-activation of thelymphocyte, and some of these methods are performed in less time thanprior methods. Furthermore, compositions that have many uses, includingtheir use in these improved methods, are provided. Some of thesecompositions are genetically modified lymphocytes that have improvedproliferative and survival qualities, including in in vitro culturing,for example in the absence of growth factors. Such genetically modifiedlymphocytes will have utility for example, as research tools to betterunderstand factors that influence T cell proliferation and survival, andfor commercial production, for example for the production of certainfactors, such as growth factors and immunomodulatory agents, that can beharvested and tested or used in commercial products.

Some embodiments provided herein are methods for performing adoptivecellular therapy that include transducing T cells and/or NK cells, thatrequire far less time ex vivo, for example, 24, 12, or 8 hours or less,and in some embodiments without prior ex vivo stimulation. These methodsare well-suited for closed system ex vivo processing of blood from asubject, and can be performed with the subject present in the same roomas and/or in some embodiments, within their line of sight of their bloodor isolated blood cells thereof at all times during performance of themethod. More specifically, the aspects and embodiments of the disclosureherein overcome problems associated with current adoptive cellulartherapies by providing methods for transducing resting T cells and/orresting NK cells, that typically utilize a pseudotyping element thatfacilitates binding and fusion of a replication incompetent recombinantretroviral particle to a resting T cell and/or a resting NK cell, tofacilitate genetic modification of the resting T cells and/or NK cellsby the replication incompetent recombinant retroviral particles.Furthermore, methods provided herein overcome problems of the art byutilizing in illustrative embodiments, a chimeric antigen receptor andone or more lymphoproliferative elements whose expression is under thecontrol of a control element, such that exposure of the subject to acompound that binds the control element, or termination of suchexposure, promotes expansion of the genetically modified T cells and/orNK cells in vivo.

As a result of these and other improvements disclosed in detail herein,in one aspect, provided herein is a method for genetically modifyingresting T cells and/or resting NK cells of a subject, such as a patienthaving a disease or disorder, wherein blood from the subject iscollected; resting T cells and/or NK cells are genetically modified bycontacting them with a replication incompetent recombinant retroviralparticle; and the genetically modified cells are reintroduced into thesubject typically within a shorter period of time than prior methods,for example within 24 hours and in some non-limiting embodiments, within12 hours and/or without further expanding the population of geneticallymodified T cells and/or NK cells ex vivo, for example such that thegenetically modified resting T cells and/or NK cells do not undergo morethan 4 cell divisions ex vivo. Thus, methods provided herein can beperformed in much less time than current CAR therapies, therebyproviding processes by which a subject can remain in a clinic for theentire time of the ex vivo steps. This facilitates performance of the exvivo steps in a closed system, which reduces the chances forcontamination and mixing of patient samples and can be performed morereadily by clinical labs.

Accordingly, FIGS. 1 and 2 provide schematic diagrams of illustrativecompositions used in methods provided herein. FIG. 1 provides a diagramof a packaging cell (100) and a replication incompetent recombinantretroviral particle, produced by such a packaging cell (200). Thepackaging cell (100) includes recombinant polynucleotides (110)incorporated into its genome that include recombinant transcriptionalelements that express retroviral proteins and various differentmembrane-bound polypeptides under the control of inducible promotersthat are regulated by transactivators, which bind and are activated byligands. These transactivators, inducible promoters, and ligands areused to induce the sequential expression and accumulation of cellmembrane-bound polypeptides that will be incorporated into the membraneof the replication incompetent recombinant retroviral particle as wellas retroviral components necessary for packaging and assembly of thereplication incompetent recombinant retroviral particles.

As a result of the sequential induced expression of the variouspolynucleotides as discussed in detail herein below, the illustrativepackaging cell (100) illustrated in FIG. 1 is produced, and can be usedin illustrative methods to produce replication incompetent recombinantretroviral particles used in methods of transfecting resting T cellsand/or NK cells ((300) in FIG. 2 ) provided herein. The packaging cell(100), in non-limiting illustrative embodiments, includes in its genomenucleic acids encoding a packageable retroviral RNA genome that includesat least some of the elements of a retroviral genome necessary forpackaging and assembly of the replication incompetent recombinantretroviral particle (as non-limiting illustrative examples, a retroviralpsi element, a retroviral gag polypeptide and a retroviral polpolypeptide).

Some membrane bound polypeptides incorporated or associated with thecell membrane of the packaging cell will become incorporated orassociated into the replication incompetent recombinant retroviralparticles, but are not encoded by the retroviral genome. For example,the packaging cell and replication incompetent recombinant retroviralparticles formed therefrom, can include a retroviral Vpx polypeptide(250), which in non-limiting illustrative examples can be expressed as amembrane associated fusion protein, for example a Src-Flag-Vpxpolypeptide; a pseudotyping element that can include a bindingpolypeptide and a fusogenic polypeptide (240), which in a non-limitingembodiment includes a Measles Virus hemagglutinin (H) polypeptide and aMeasles Virus fusion (F) polypeptide, or cytoplasmic domain deletionvariants thereof; optionally, one or more activation elements (210,220), which in a non-limiting embodiment includes a membrane-boundpolypeptide capable of binding to CD3 and a membrane-bound polypeptidecapable of binding to CD28; and/or optionally a membrane-bound cytokine(230), a non-limiting embodiment of which is a fusion polypeptide thatincludes IL-7 fused to DAF, or a fragment thereof. Various otherspecific types of these membrane bound polypeptides are provided herein.

As a result of the sequential expression of the transcriptional elementsby the packaging cell, a replication incompetent recombinant retroviralparticle is produced. The RNA retroviral genome inside of and typicallyintegrated into the genome of the packaging cell that becomes the genomeof the replication incompetent recombinant retroviral particle, includesretroviral components (as non-limiting illustrative examples, retroviralGag and Pol polynucleotides) that are necessary for retroviralproduction, infection and integration into the genome of a host cell,which is typically a resting T cell and/or NK cell. Furthermore, theretroviral genome furthermore includes polynucleotides encoding one ortypically two engineered signaling polypeptides provided herein. One ofthe engineered signaling polypeptides typically encodes at least onelymphoproliferative element (in non-limiting examples a constitutiveinterleukin 7 receptor mutant) and the other engineered signalingpolypeptide typically encodes a chimeric antigen receptor.

The replication incompetent recombinant retroviral particle, (200) isthen used to transduce a resting T cell and/or resting NK cell (300) inmethods provided herein. As shown in FIG. 2 , after the resting T celland/or NK cell (300) is contacted with the replication incompetentrecombinant retroviral particle (200), membrane polypeptides discussedabove on the surface of the replication incompetent recombinantretroviral particle bind to receptors and/or ligands on the surface ofthe resting T cell and/or NK cell (300). For example, the pseudotypingelement, which as indicated above can include a binding polypeptide thatbinds to molecules on the surface of resting T cells and/or resting NKcells and a fusogenic polypeptide, facilitates the binding and fusion ofreplication incompetent recombinant retroviral particle (200) to the Tcell and/or NK cell membrane. The activation element(s) (210, 220)activate the resting T cell and/or NK cell (300) by engaging the T-cellreceptor complex, a process which occurs over the time course of thecontacting or an incubation thereafter. Furthermore, the membrane-boundcytokines (230) can be present on the surface of replication incompetentrecombinant retroviral particle and bind cytokine receptors (310) on thesurface of the resting T cell and/or NK cell (300), thus furtherpromoting binding and activation. Thus, not to be limited by theory, inillustrative embodiments provided herein, as a result of one or more ofthese replication incompetent recombinant retroviral particles (200)components, ex vivo stimulation or activation by an element that is notalready in or on the replication incompetent recombinant retroviralparticle (200) is not required. This in turn, helps to cut down the exvivo time that is required for completion of the methods in theseillustrative methods provided herein.

Upon binding to the T cell and/or NK cell (200), the replicationincompetent recombinant retroviral particle then fuses with the T celland/or NK cell (300), and polypeptides and nucleic acids in thereplication incompetent recombinant retroviral particle enter the T celland/or NK cell (300). As indicated above, one of these polypeptides inthe replication incompetent recombinant retroviral particle is the Vpxpolypeptide (250). The Vpx polypeptide (250) binds to and induces thedegradation of the SAMHD1 restriction factor (350), which degrades freedNTPs in the cytoplasm. Thus, the concentration of free dNTPs in thecytoplasm increases as Vpx degrades SAMHD1, and reverse transcriptionactivity is increased, thus facilitating reverse transcription of theretroviral genome and integration into the T cell and/or NK cell genome.

After integration of the retroviral genome into the T cell and/or NKcell (200), the T cell and/or NK cell genome includes nucleic acidsencoding the signaling polypeptide encoding the lymphoproliferativeelement (370) and optionally the signaling polypeptide encoding the CAR(360). Expression of the lymphoproliferative element and optionally theCAR are under the control of a control element. Exposure to a compoundthat binds the control element, which can occur in vitro or in vivo byadministering it to a subject whose T cell and/or NK cell (300) wastransduced, promotes proliferation of the T cell and/or NK cell (300) invitro or in vivo by expressing the lymphoproliferative element andoptionally as a result of expression of the CAR and binding of the CARto its target cell. Thus, T cells and/or NK cells that are transducedwith replication incompetent recombinant retroviral particles herein,have one or more signals that drive proliferation and/or inhibit celldeath, which in turn in illustrative embodiments, avoids therequirements of prior methods to lymphodeplete a host before returningtransduced T cells and/or NK cells back into the subject. This in turn,in illustrative embodiments, further reduces the requirement for days ofprocessing before transduced T cells and/or NK cells are reintroducedinto a subject. Thus, in illustrative embodiments, no more than 36hours, 24 hours, 12 hours, or in some instances even 8 hours, of time isrequired from collection of blood from the subject to reintroduction ofthe blood to the subject, which fundamentally changes the CAR-T processfrom prior methods. Furthermore, the control element provides one of thesafety mechanisms provided herein as well. For example, ceasingadministration of the compound can down-regulate or even terminateexpression of the lymphoproliferative element and optionally the CAR,thus ending a proliferation and/or survival signal to the transduced Tcell and/or NK cell and its progeny.

Methods for Transducing and/or Genetically Modifying Lymphocytes

Provided herein in certain aspects, is a method of transducing and/orgenetically modifying a lymphocyte, typically a T cell and/or an NKcell, and typically a resting T cell and/or resting NK cell, thatincludes contacting the lymphocyte with a replication incompetentrecombinant retroviral particle, wherein the replication incompetentrecombinant retroviral particle typically comprises a pseudotypingelement on its surface, wherein said contacting (and incubation undercontacting conditions) facilitates transduction of the resting T celland/or NK cell by the replication incompetent recombinant retroviralparticle, thereby producing the genetically modified T cell and/or NKcell. The pseudotyping element is typically capable of binding theresting T cell and/or NK cell and typically facilitating membrane fusionon its own or in conjunction with other protein(s) of the replicationincompetent recombinant retroviral particles.

In some embodiments, the replication incompetent recombinant retroviralparticle can further include an activation element, which can be anyactivation element provided herein. In illustrative embodiments, theactivation element can be anti-CD3, such as anti-CD3 scFv, or anti-CD3scFvFc. In some embodiments, the contacting can be performed for between1 and 24 hours, for example, between 1 and 12 hours, or between 1 and 6hours. In some embodiments, the contacting can be performed for lessthan 24 hours, for example, less than 12 hours, less than 8 hours, orless than 4 hours. A packing cell, and in illustrative embodiments apackaging cell line, and in particularly illustrative embodiments apackaging cell provided in certain aspects herein, is used to producethe replication incompetent recombinant retroviral particle. Inexemplary embodiments of such methods, the genetically modified T cellor NK cell is capable of survival in ex vivo culture for at least 7, 14,21, 28, 35, 42, or 60 days in the absence of a target antigen recognizedby the CAR and in the absence of cytokines such as IL-2, IL-15, or IL-7.In exemplary embodiments of such methods, the genetically modified Tcell or NK cell is capable of engraftment in vivo in mice and/orenrichment in vivo in mice for at least 7, 14, or 28 days. A skilledartisan will recognize that such mice may be treated or otherwisegenetically modified so that any immunological differences between thegenetically modified T cell and/or NK cell do not result in an immuneresponse being elicited in the mice against any component of thelymphocyte transduced by the replication incompetent recombinantretroviral particle. In some embodiments, the packaging cell line can bea suspension cell line. In illustrative embodiments, the packaging cellline can be grown in serum-free media. In some embodiments, thelymphocyte can be from a subject. In illustrative embodiments, thelymphocyte can be from blood of a subject.

Further embodiments of any of the above aspects for transducing and/orgenetically modifying a lymphocyte, for example an NK cell or inillustrative embodiments, a T cell, can include any of the embodimentsof replication incompetent recombinant retroviral particles,lymphoproliferative elements, CARs, pseudotyping elements, riboswitches,activation elements, membrane-bound cytokines, miRNAs, and/or otherelements disclosed herein, and can be combined with methods herein forproducing retroviral particles using a packaging cell. In certainillustrative embodiments, the retroviral particle is a lentiviralparticle. Such a method for transducing a T cell and/or NK cell can beperformed in vitro or ex vivo. A skilled artisan will recognize thatdetails provided herein for transducing and/or genetically modifying Tcell(s) and/or NK cell(s) can apply to any aspect that includes suchstep(s), including aspects that are directed to methods for transducingand/or genetically modifying a lymphocyte such as T cell(s) and/or NKcell(s).

Accordingly, provided in one aspect herein is a method for transducing(and/or genetically modifying) lymphocytes, typically resting T cellsand/or resting NK cells from isolated blood, comprising:

-   -   A. collecting blood from a subject;    -   B. isolating peripheral blood mononuclear cells (PBMCs)        comprising resting T cells and/or resting NK cells; and    -   C. contacting the resting T cells and/or resting NK cells of the        subject ex vivo, with replication incompetent recombinant        retroviral particles, wherein the replication incompetent        recombinant retroviral particles comprise a pseudotyping element        on their surface that is capable of binding a resting T cell        and/or resting NK cell and facilitating membrane fusion of the        replication incompetent recombinant retroviral particles        thereto, wherein said contacting facilitates transduction of at        least 5% of the resting T cells and/or resting NK cells by the        replication incompetent recombinant retroviral particles,        thereby producing genetically modified T cells and/or NK cells,        thereby transducing resting T cells and/or NK cells.

In some embodiments of any method herein that includes a step oftransducing T cells and/or NK cells, the cells can be contacted with aretroviral particle without prior activation. In some embodiments of anymethod herein that includes a step of transducing T cells and/or NKcells, the T cells and/or NK cells have not been incubated on asubstrate that adheres to monocytes for more than 4 hours in oneembodiment, or for more than 6, hours in another embodiment, or for morethan 8 hours in another embodiment before the transduction. In oneillustrative embodiment, the T cells and/or NK cells have been incubatedovernight on an adherent substrate to remove monocytes before thetransduction. In another embodiment, the method can include incubatingthe T cells and/or NK cells on an adherent substrate that bindsmonocytes for no more than 30 minutes, 1 hour, or 2 hours before thetransduction. In another embodiment, the T cells and/or NK cells areexposed to no step of removing monocytes by an incubation on an adherentsubstrate before said transduction step. In another embodiment, the Tcells and/or NK cells are not incubated with or exposed to a bovineserum, such as a cell culturing bovine serum, for example fetal bovineserum before or during the transduction.

Accordingly, provided in another aspect herein is a method forgenetically modifying or transducing a lymphocyte of a subject, inillustrative embodiments, a T cell and/or and NK cell or a population ofT cells or NK cells, that includes contacting the T cell(s) and/or NKcell(s) of, typically of a subject ex vivo, with a replicationincompetent recombinant retroviral particle comprising in its genome apolynucleotide comprising one or more nucleic acid sequences operativelylinked to a promoter active in T cells and/or NK cells, wherein a firstnucleic acid sequence of the one or more nucleic acid sequences encodestwo or more inhibitory RNA molecules directed against one or more RNAtargets and a second nucleic acid sequence of the one or more nucleicacid sequences encodes a chimeric antigen receptor (CAR) comprising anantigen-specific targeting region (ASTR), a transmembrane domain, and anintracellular activating domain, wherein said contacting facilitatestransduction of the, or at least some of the resting T cells and/or NKcells by the replication incompetent recombinant retroviral particle,thereby producing a genetically modified T cell and/or NK cell.

Provided herein in another aspect is a method for genetically modifyingor transducing a lymphocyte (e.g. a T cell or an NK cell) or apopulation thereof, of a subject, comprising contacting the lymphocyte(e.g. the T cell or NK cell) or a population thereof, of the subject exvivo, with a replication incompetent recombinant retroviral particlecomprising in its genome a polynucleotide comprising one or more nucleicacid sequences operatively linked to a promoter active in lymphocytes(e.g. T cells and/or NK cells), wherein a first nucleic acid sequence ofthe one or more nucleic acid sequences encodes one or more (e.g. two ormore) inhibitory RNA molecules directed against one or more RNA targetsand a second nucleic acid sequence of the one or more nucleic acidsequences encodes a chimeric antigen receptor (CAR) comprising anantigen-specific targeting region (ASTR), a transmembrane domain, and anintracellular activating domain, wherein said contacting facilitatesgenetic modification and/or transduction of the lymphocyte (e.g. T cellor NK cell), or at least some of the lymphocytes (e.g. T cells and/or NKcells) by the replication incompetent recombinant retroviral particle,thereby producing a genetically modified and/or transduced lymphocyte(e.g. T cell and/or NK cell).

In some embodiments of the method provided immediately above, thegenetically modified and/or transduced lymphocyte (e.g. T cell and/or NKcell) or population thereof, is introduced into the subject. In someembodiments, the genetically modified and/or transduced lymphocyte (e.g.T cell and/or NK cell) or population thereof, undergoes 4 or fewer celldivisions ex vivo prior to being introduced or reintroduced into thesubject. In some embodiments, the lymphocyte(s) are resting T cellsand/or resting NK cells that are in contact with the replicationincompetent recombinant retroviral particles for between 1 hour and 12hours. In some embodiments, no more than 8 hours pass between the timeblood is collected from the subject and the time the geneticallymodified T cells and/or NK cells are reintroduced into the subject. Insome embodiments, all steps after the blood is collected and before theblood is reintroduced, are performed in a closed system in which aperson monitors the closed system throughout the processing.

In any of the method aspects provided immediately above that include apolynucleotide comprising one or more nucleic acid sequences operativelylinked to a promoter active in T cells and/or NK cells, wherein a firstnucleic acid sequence of the one or more nucleic acid sequences encodesone or more (e.g. two or more) inhibitory RNA molecules directed againstone or more RNA targets, and a second nucleic acid sequence of the oneor more nucleic acid sequences encodes a chimeric antigen receptor (CAR)comprising an antigen-specific targeting region (ASTR), a transmembranedomain, and an intracellular activating domain, the polynucleotide mayfurther include a third nucleic acid sequence that encodes at least onelymphoproliferative element that is not an inhibitory RNA molecule. Insome embodiments, the lymphoproliferative element can be a cytokine orcytokine receptor polypeptide, or a fragment thereof comprising asignaling domain. In some embodiments, the lymphoproliferative elementis constitutively active. In certain embodiments, thelymphoproliferative element can be an IL-7 receptor or a fragmentthereof. In illustrative embodiments, the lymphoproliferative elementcan be a constitutively active IL-7 receptor or a constitutively activefragment thereof.

In any of the method aspects provided immediately above that include apolynucleotide comprising one or more nucleic acid sequences operativelylinked to a promoter active in T cells and/or NK cells, wherein a firstnucleic acid sequence of the one or more nucleic acid sequences encodesone or more (e.g. two or more) inhibitory RNA molecules directed againstone or more RNA targets, an inhibitory RNA molecule can in someembodiments include a 5′ strand and a 3′ strand that are partially orfully complementary to one another, wherein said 5′ strand and said 3′strand are capable of forming an 18-25 nucleotide RNA duplex. In someembodiments, the 5′ strand can be 18, 19, 20, 21, 22, 23, 24, or 25nucleotides in length, and the 3′ strand can be 18, 19, 20, 21, 22, 23,24, or 25 nucleotides in length. In some embodiments, the 5′ strand andthe 3′ strand can be the same or different lengths. In some embodiments,the RNA duplex can include one or more mismatches. In alternateembodiments, the RNA duplex has no mismatches.

In any of the method aspects provided immediately above that include apolynucleotide comprising one or more nucleic acid sequences operativelylinked to a promoter active in T cells and/or NK cells, wherein a firstnucleic acid sequence of the one or more nucleic acid sequences encodesone or more (e.g. two or more) inhibitory RNA molecules directed againstone or more RNA targets, an inhibitory RNA molecule can be a miRNA or anshRNA. In some embodiments, the inhibitory molecule can be a precursorof a miRNA, such as for example, a Pri-miRNA or a Pre-miRNA, or aprecursor of an shRNA. In some embodiments, the inhibitory molecule canbe an artificially derived miRNA or shRNA. In other embodiments, theinhibitory RNA molecule can be a dsRNA (either transcribed orartificially introduced) that is processed into an siRNA or the siRNAitself. In some embodiments, the inhibitory RNA molecule can be a miRNAor shRNA that has a sequence that is not found in nature, or has atleast one functional segment that is not found in nature, or has acombination of functional segments that are not found in nature. Inillustrative embodiments, at least one or all of the inhibitory RNAmolecules are miR-155.

In any of the method aspects provided immediately above that include apolynucleotide comprising one or more nucleic acid sequences operativelylinked to a promoter active in T cells and/or NK cells, wherein a firstnucleic acid sequence of the one or more nucleic acid sequences encodesone or more (e.g. two or more) inhibitory RNA molecules directed againstone or more RNA targets, an inhibitory RNA molecule, in someembodiments, can comprises from 5′ to 3′ orientation: a 5′ arm, a 5′stem, a loop, a 3′ stem that is partially or fully complementary to said5′ stem, and a 3′ arm. In some embodiments, at least one of two or moreinhibitory RNA molecules has this arrangement. In other embodiments, allof two or more inhibitory molecules have this arrangement. In someembodiments, the 5′ stem can be 18, 19, 20, 21, 22, 23, 24 or 25nucleotides in length. In some embodiments, the 3′ stem can be 18, 19,20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments,the loop can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 2 5, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, or 40 nucleotides in length. In some embodiments, the 5′arm, 3′ arm, or both, are derived from a naturally occurring miRNA. Insome embodiments, the 5′ arm, 3′ arm, or both, are derived from anaturally occurring miRNA is selected from the group consisting of:miR-155, miR-30, miR-17-92, miR-122, and miR-21. In illustrativeembodiments, the 5′ arm, 3′ arm, or both are derived from miR-155. Insome embodiments, the 5′ arm, 3′ arm, or both are derived from Musmusculus miR-155 or Homo sapiens miR-155. In some embodiments, the 5′arm has the sequence set forth in SEQ ID NO:256 or is a functionalvariant thereof, such as, for example, a sequence that is the samelength as SEQ ID NO:256, or 95%, 90%, 85%, 80%, 75%, or 50% as long asSEQ ID NO: 256 or is 100 nucleotides or less, 95 nucleotides or less, 90nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75nucleotides or less, 70 nucleotides or less, 65 nucleotides or less, 60nucleotides or less, 55 nucleotides or less, 50 nucleotides or less, 45nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30nucleotides or less, or 25 nucleotides or less; and is at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ IDNO:256. In some embodiments, the 3′ arm has the sequence set forth inSEQ ID NO:260 or is a functional variant thereof, such as, for example,the same length as SEQ ID NO:260, or 95%, 90%, 85%, 80%, 75%, or 50% aslong as SEQ ID NO: 260 or is a sequence that is 100 nucleotides or less,95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less,80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less,65 nucleotides or less, 60 nucleotides or less, 55 nucleotides or less,50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less,35 nucleotides or less, 30 nucleotides or less, or 25 nucleotides orless; and is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or95% identical to SEQ ID NO:260. In some embodiments, the 3′ armcomprises nucleotides 221-283 of the Mus musculus BIC.

In any of the method aspects provided immediately above that include apolynucleotide comprising one or more nucleic acid sequences operativelylinked to a promoter active in T cells and/or NK cells, wherein a firstnucleic acid sequence of the one or more nucleic acid sequences encodestwo or more inhibitory RNA molecules directed against one or more RNAtargets, the two or more inhibitory RNA molecules, in some embodiments,can be positioned in the first nucleic acid sequence in series. In someembodiments, the inhibitory RNA molecules can be adjoined to one anothereither directly or indirectly by non-functional linker sequence(s). Insome embodiments, the linker sequences can be between 5 and 120nucleotides in length, or between 10 and 40 nucleotides in length.

In any of the method aspects provided immediately above that include apolynucleotide comprising one or more nucleic acid sequences operativelylinked to a promoter active in T cells and/or NK cells, wherein a firstnucleic acid sequence of the one or more nucleic acid sequences encodestwo or more inhibitory RNA molecules directed against one or more RNAtargets, in some embodiments, the first nucleic acid sequence encodestwo to four inhibitory RNA molecules. In illustrative embodiments,between 2 and 10, 2 and 8, 2 and 6, 2 and 5, 2 and 4, 3 and 5, or 3 and6 inhibitory RNA molecules are included in the first nucleic acidsequence. In an illustrative embodiment, four inhibitory RNA moleculesare included in the first nucleic acid sequence.

In any of the method aspects provided immediately above that include apolynucleotide comprising one or more nucleic acid sequences operativelylinked to a promoter active in T cells and/or NK cells, wherein a firstnucleic acid sequence of the one or more nucleic acid sequences encodesone or more (e.g. two or more) inhibitory RNA molecules directed againstone or more RNA targets, the one or more (e.g. two or more) inhibitoryRNA molecules can be in an intron. In some embodiments, the intron is ina promoter. In illustrative embodiments, the intron is EF-1alpha intronA. In some embodiments, the intron is adjacent to and downstream of apromoter, which in illustrative embodiments, is inactive in a packagingcell used to produce the replication incompetent recombinant retroviralparticle.

In any of the method aspects provided immediately above that include apolynucleotide comprising one or more nucleic acid sequences operativelylinked to a promoter active in T cells and/or NK cells, wherein a firstnucleic acid sequence of the one or more nucleic acid sequences encodestwo or more inhibitory RNA molecules directed against one or more RNAtargets, the two or more inhibitory RNA molecules, in some embodiments,can be directed against different targets. In an alternate embodiment,the two or more inhibitory RNA molecules are directed against the sametarget. In some embodiments, the RNA targets are mRNAs transcribed fromgenes that are expressed by T cells such as but not limited to PD-1(prevent inactivation); CTLA4 (prevent inactivation); TCRa(safety—prevent autoimmunity); TCRb (safety—prevent autoimmunity); CD3Z(safety—prevent autoimmunity); SOCS1 (prevent inactivation); SMAD2(prevent inactivation); a miR-155 target (promote activation); IFN gamma(reduce CRS); cCBL (prolong signaling); TRAIL2 (prevent death); PP2A(prolong signaling); ABCG1 (increase cholesterol microdomain content bylimiting clearance of cholesterol). In some embodiments, the RNA targetsare mRNAs transcribed from genes that encode components of the T cellreceptor (TCR) complex. In some embodiments, at least one of the two ormore of inhibitory RNA molecules can decrease expression of T cellreceptors, in illustrative embodiments, one or more endogenous T cellreceptor(s) of a T cell. In certain embodiments, the RNA target can bemRNA transcribed from the endogenous TCRα or TCRβ gene of the T cellwhose genome comprises the first nucleic acid sequence encoding the oneor more miRNAs. In illustrative embodiments, the RNA target is mRNAtranscribed from the TCRα gene.

In any of the method aspects provided immediately above that include apolynucleotide comprising one or more nucleic acid sequences operativelylinked to a promoter active in T cells and/or NK cells, wherein a firstnucleic acid sequence of the one or more nucleic acid sequences encodesone or more (e.g. two or more) inhibitory RNA molecules directed againstone or more RNA targets, and a second nucleic acid sequence of the oneor more nucleic acid sequences encodes a chimeric antigen receptor (CAR)comprising an antigen-specific targeting region (ASTR), a transmembranedomain, and an intracellular activating domain, in some embodiments, theCAR is a microenvironment restricted biologic (MRB)-CAR. In otherembodiments, the ASTR of the CAR binds to a tumor associated antigen. Inother embodiments, the ASTR of the CAR is a microenvironment-restrictedbiologic (MRB)-ASTR.

In any of the method aspects provided immediately above that include apolynucleotide comprising one or more nucleic acid sequences operativelylinked to a promoter active in T cells and/or NK cells, wherein a firstnucleic acid sequence of the one or more nucleic acid sequences encodesone or more (e.g. two or more) inhibitory RNA molecules directed againstone or more RNA targets, and a second nucleic acid sequence of the oneor more nucleic acid sequences encodes a chimeric antigen receptor (CAR)comprising an antigen-specific targeting region (ASTR), a transmembranedomain, and an intracellular activating domain, and in some instances athird nucleic acid sequence of the one or more nucleic acid sequencesthat encodes at least one lymphoproliferative element that is not aninhibitory RNA molecule, in some embodiments, any or all of the firstnucleic acid sequence, second nucleic acid sequence, and third nucleicacid sequence is operably linked to a riboswitch. In some embodiments,the riboswitch is capable of binding a nucleoside analog. In someembodiments, the nucleoside analog is an antiviral drug.

In some embodiments, methods are provided for activating and/orgenetically modifying, and typically transducing resting T cells or NKcells, in illustrative embodiments resting T cells, by contacting thecells with a retroviral particle disclosed herein and soluble anti-CD3antibodies at 25-200, 50-150, 75-125, or 100 ng/ml. In illustrativeembodiments, such methods are performed without prior activation, andcan be carried out, for example, for 8 hours or less, 4 hours or less,or between 2 and 8 hours, 2 and 4 hours, or between 2 and 3 hours.

In certain aspects, provided herein are methods for performing adoptivecell therapy on a subject, As an illustrative example, the method caninclude the following:

-   -   A. collecting blood from a subject;    -   B. isolating peripheral blood mononuclear cells (PBMCs)        comprising resting T cells and/or resting NK cells;    -   C. contacting the resting T cells and/or resting NK cells of the        subject ex vivo, with replication incompetent recombinant        retroviral particles, wherein the replication incompetent        recombinant retroviral particles comprise a pseudotyping element        on their surface that is capable of binding a resting T cell        and/or NK cell and facilitating membrane fusion of the        replication incompetent recombinant retroviral particles        thereto, wherein said contacting facilitates transduction of the        resting T cells and/or NK cells by the replication incompetent        recombinant retroviral particles, thereby producing genetically        modified T cells and/or NK cells; and    -   D. reintroducing the genetically modified cells into the subject        within 36, 24, 12, or even 8 hours of collecting blood from the        subject, thereby performing adoptive cell therapy in the        subject.

In some aspects provided herein, methods with similar steps are referredto as methods for genetically modifying and expanding lymphocytes of asubject. A skilled artisan will understand that the discussion herein asit applies to methods and compositions for performing adoptive celltherapy apply to methods for genetically modifying and expandinglymphocytes of a subject as well.

Typically, the adoptive cell therapy methods of the present disclosureare carried out by autologous transfer, in which the cells are isolatedand/or otherwise prepared from the subject who is to receive the celltherapy, or from a sample derived from such a subject. Thus, in someaspects, the cells are derived from a subject, e.g., patient, in need ofa treatment and the cells, following isolation and processing areadministered to the same subject. In some embodiments of the methods andcompositions disclosed herein, a subject having a disease or disorderenters a medical facility where the subject's blood is drawn using knownmethods, such as venipuncture. In certain embodiments, the volume ofblood drawn from a subject is between 10, 15, 20, 25, 30, 35, 40, 50,75, or 100 ml on the low end of the range and 200, 250, 300, 350, 400,500, 750, 1000, 2000, or 2500 ml on the high end of the range. In someembodiments, between 10 and 400 ml are drawn from the subject. In someembodiments, between 20 and 250 ml of blood are drawn from the subject.In some embodiments, the blood is fresh when it is processed. In any ofthe embodiments disclosed herein, fresh blood can be blood that waswithdrawn from a subject less than 15, 30, 45, 60, 90, 120, 150, or 180minutes prior. In some embodiments, the blood is processed in themethods provided herein without storage.

Contact between the T cells and/or NK cells and the replicationincompetent recombinant retroviral particles typically facilitatestransduction of the T cells and/or NK cells by the replicationincompetent recombinant retroviral particles. Throughout thisdisclosure, a transduced T cell and/or NK cell includes progeny of exvivo transduced cells that retain at least some of the nucleic acids orpolynucleotides that are incorporated into the cell during the ex vivotransduction. In methods herein that recite “reintroducing” a transducedcell, it will be understood that such cell is typically not in atransduced state when it is collected from the blood of a subject. Asubject in any of the aspects disclosed herein can be for example, ananimal, a mammal, and in illustrative embodiments a human.

Not to be limited by theory, in non-limiting illustrative methods, thedelivery of a polynucleotide encoding a lymphoproliferative element,such as an IL7 constitutively active mutant, to a resting T cell and/orNK cell ex vivo, which can integrate into the genome of the T cell or NKcell, provides that cell with a driver for in vivo expansion without theneed for lymphodepleting the host. Thus, in illustrative embodiments,the subject is not exposed to a lymphodepleting agent within 1, 2, 3, 4,5, 6, 7, 10, 14, 21, or 28 days, or within 1 month, 2 months, 3 monthsor 6 months of performing the contacting, during the contacting, and/orwithin 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 days, or within 1 month, 2months, 3 months or 6 months after the modified T cells and/or NK cellsare reintroduced back into the subject. Furthermore, in non-limitingillustrative embodiments, methods provided herein can be performedwithout exposing the subject to a lymphodepleting agent during a stepwherein a replication incompetent recombinant retroviral particle is incontact with resting T cells and/or resting NK cells of the subjectand/or during the entire ex vivo method.

Hence, methods of expanding genetically modified T cells and/or NK cellsin a subject in vivo is a feature of some embodiments of the presentdisclosure. In illustrative embodiments, such methods are ex vivopropagation-free or substantially propagation-free.

This entire method/process from blood draw from a subject toreintroduction of blood back into the subject after ex vivo transductionof T cells and/or NK cells, in non-limiting illustrative embodimentsherein, can occur over a time period less than 48 hours, less than 36hours, less than 24 hours, less than 12 hours, less than 11 hours, lessthan 10 hours, less than 9 hours, less than 8 hours, less than 7 hours,less than 6 hours, less than 5 hours, less than 4 hours, less than 3hours, 2 hours, or less than 2 hours. In other embodiments, the entiremethod/process from blood draw/collection from a subject toreintroduction of blood back into the subject after ex vivo transductionof T cells and/or NK cells, in non-limiting illustrative embodimentsherein, occurs over a time period between 1 hour and 12 hours, orbetween 2 hours and 8 hours, or between 1 hour and 3 hours, or between 2hours and 4 hours, or between 2 hours and 6 hours, or between 4 hoursand 12 hours, or between 4 hours and 24 hours, or between 8 hours and 24hours, or between 8 hours and 36 hours, or between 8 hours and 48 hours,or between 12 hours and 24 hours, or between 12 hours and 36 hours, orbetween 12 hours and 48 hours, or over a time period between 15, 30, 60,90, 120, 180, and 240 minutes on the low end of the range, and 120, 180,and 240, 300, 360, 420, and 480 minutes on the high end of the range. Inother embodiments, the entire method/process from blood draw/collectionfrom a subject to reintroduction of blood back into the subject after exvivo transduction of T cells and/or NK cells, occurs over a time periodbetween 1, 2, 3, 4, 6, 8, 10, and 12 hours on the low end of the range,and 8, 9, 10, 11, 12, 18, 24, 36, or 48 hours on the high end of therange. In some embodiments, the genetically modified T cells and/or NKcells are separated from the replication incompetent recombinantretroviral particles after the time period in which contact occurs.

In some embodiments of any method herein that includes a step of bloodcollection and a step of transduction of lymphocytes, in illustrativeembodiments T cells and/or NK cells, including resting T cell and NKcells, the method from blood collection through transduction of T cellsand/or NK cells does not include a step of removing monocytes by anincubation on an adherent substrate of more than 4 hours in oneembodiment, or for more than 6, hours in another embodiment, or for morethan 8 hours in another embodiment. In one illustrative embodiment, themethod from blood collection through transduction of T cells and/or NKcells does not include an overnight incubation on an adherent substrateto remove monocytes. In another embodiment, the method from bloodcollection through transduction of T cells and/or NK cells includes astep of removing monocytes by an incubation on an adherent substrate forno more than 30 minutes, 1 hour, or 2 hours. In another embodiment, themethod from blood collection from a subject through transduction oflymphocytes, in illustrative embodiments T cells and/or NK cells,including resting T cells and/or NK cells, include no step of removingmonocytes by an incubation on an adherent substrate. In anotherembodiment, In another embodiment, the method from blood collection froma subject through transduction of lymphocytes, in illustrativeembodiments T cells and/or NK cells, including resting T cells and/or NKcells, includes, the T cells and/or NK cells are not incubated with orexposed to a bovine serum such as a cell culturing bovine serum, forexample fetal bovine serum during the method.

In some embodiments of any method herein that includes a step of bloodcollection and a step of transduction of lymphocytes, in illustrativeembodiments T cells and/or NK cells, including resting T cell and NKcells, the method from blood collection from a subject throughreintroduction of T cells and/or NK cells into the subject does notinclude a step of removing monocytes by an incubation on an adherentsubstrate of more than 4 hours in one embodiment, or for more than 6,hours in another embodiment, or for more than 8 hours in anotherembodiment. In one illustrative embodiment, the method from bloodcollection from a subject through reintroduction of T cells and/or NKcells into the subject does not include an overnight incubation on anadherent substrate to remove monocytes. In another embodiment, themethod from blood collection from a subject through reintroduction of Tcells and/or NK cells into the subject includes a step of removingmonocytes by an incubation on an adherent substrate for no more than 30minutes, 1 hour, or 2 hours. In another embodiment, the method fromblood collection from a subject through reintroduction of T cells and/orNK cells into the subject includes no step of removing monocytes by anincubation on an adherent substrate. In another embodiment, the methodfrom blood collection from a subject through reintroduction of T cellsand/or NK cells into the subject, the T cells and/or NK cells are notincubated with or exposed to a bovine serum, such as a cell culturingbovine serum, for example fetal bovine serum during the method.

Because methods provided herein for adoptive cell therapy and relatedmethods for modifying resting T cells and/or resting NK cells ex vivobefore expanding them in vivo, can be performed in significantly lesstime than prior methods, fundamental improvements in patient care andsafety as well as product manufacturability are made possible.Therefore, such processes are expected to be favorable in the view ofregulatory agencies responsible for approving such processes whencarried out in vivo for therapeutic purposes. For example, the subjectin non-limiting examples, can remain in the same building (e.g. infusionclinic) or room as the instrument processing their blood or sample forthe entire time that the sample is being processed before modified Tcells and/or NK cells are reintroduced into the patient. In non-limitingillustrative embodiments, a subject remains within line of site and/orwithin 100, 50, 25, or 12 feet or arm's distance of their blood or cellsthat are being processed, for the entire method/process from blooddraw/collection from the subject to reintroduction of blood to thesubject after ex vivo transduction of T cells and/or NK cells. In othernon-limiting illustrative embodiments, a subject remains awake and/or atleast one person can continue to monitor the blood or cells of thesubject that are being processed, throughout and/or continuously for theentire method/process from blood draw/collection from the subject toreintroduction of blood to the subject after ex vivo transduction of Tcells and/or NK cells. Because of improvements provided herein, theentire method/process for adoptive cell therapy and/or for transducingresting T cells and/or NK cells from blood draw/collection from thesubject to reintroduction of blood to the subject after ex vivotransduction of T cells and/or NK cells can be performed with continuousmonitoring by a human. In other non-limiting illustrative embodiments,at no point the entire method/process from blood draw/collection fromthe subject to reintroduction of blood to the subject after ex vivotransduction of T cells and/or NK cells, are blood cells incubated in aroom that does not have a person present. In other non-limitingillustrative embodiments, the entire method/process from blooddraw/collection from the subject to reintroduction of blood to thesubject after ex vivo transduction of T cells and/or NK cells, isperformed next to the subject and/or in the same room as the subjectand/or next to the bed or chair of the subject. Thus, sample identitymix-ups can be avoided, as well as long and expensive incubations overperiods of days or weeks. This is further provided by the fact thatmethods provided herein are readily adaptable to closed and automatedblood processing systems, where a blood sample and its components thatwill be reintroduced into the subject, only make contact withdisposable, single-use components.

Methods for performing adoptive cell therapy provided herein, typicallyinclude 1) methods of transducing lymphocytes, such as T cell(s) or NKcell(s), which in illustrative embodiments are resting T cell(s) and/orNK cell(s), and/or include 2) methods for genetically modifying alymphocyte such as T cell(s) and/or an NK cell(s), which in illustrativeembodiments are resting T cell(s) and/or NK cell(s), both (1 and 2) ofwhich themselves each form distinct aspects of the present disclosure.Such methods can be performed with or without other steps identifiedherein for performing adoptive cell therapy. In methods for adoptivecell therapy and any method provided herein that include transducingresting T cells and/or resting NK cells ex vivo, typically,neutrophils/granulocytes are separated away from the blood cells beforethe cells are contacted with replication incompetent recombinantretroviral particles. In some embodiments, peripheral blood mononuclearcells (PBMCs) including peripheral blood lymphocytes (PBLs) such as Tcell and/or NK cells, are isolated away from other components of a bloodsample using for example, apheresis, and/or density gradientcentrifugation. In some embodiments, neutrophils are removed beforePBMCs and/or T cells and/or NK cells are processed, contacted with areplication incompetent recombinant retroviral particle, transduced, ortransfected. With reference to the subject to be treated, the cells maybe allogeneic and/or autologous.

As non-limiting examples, in some embodiments for methods of transducingor genetically modifying herein, PBMCs are isolated using a Sepax orSepax 2 cell processing system (BioSafe). In some embodiments, the PBMCsare isolated using a CliniMACS Prodigy cell processor (Miltenyi Biotec).In some embodiments, an automated apheresis separator is used whichtakes blood from the subject, passes the blood through an apparatus thatsorts out a particular cell type (such as, for example, PBMCs), andreturns the remainder back into the subject. Density gradientcentrifugation can be performed after apheresis. In some embodiments,the PBMCs are isolated using a leukoreduction filter device. In someembodiments, magnetic bead activated cell sorting is then used forpurifying a specific cell population from PBMCs, such as, for example,PBLs or a subset thereof, according to a cellular phenotype (i.e.positive selection). Other methods for purification can also be used,such as, for example, substrate adhesion, which utilizes a substratethat mimics the environment that a T cell encounters during recruitment,allowing them to adhere and migrate, or negative selection, in whichunwanted cells are targeted for removal with antibody complexes thattarget the unwanted cells. In some embodiments, red blood cell rosettingcan be used to purify cells.

In some illustrative embodiments of any of the relevant aspects herein,the PBLs include T cells and/or NK cells. The T cells and/or NK cellsthat are contacted by replication incompetent recombinant retroviralparticles of the present disclosure during certain embodiments herein,for example in methods of modifying lymphocytes and methods ofperforming adoptive cellular therapy, are mainly resting T cells. Insome embodiments, the T cells and/or NK cells consist of between 95 and100% resting cells (Ki-67⁻). In some embodiments, the T cell and/or NKcells that are contacted by replication incompetent recombinantretroviral particles include between 90, 91, 92, 93, 94, and 95% restingcells on the low end of the range and 96, 97, 98, 99, or 100% restingcells on the high end of the range. In some embodiments, the T cellsand/or NK cells include naïve cells.

In some embodiments of the methods and compositions disclosed herein, Tcells and/or NK cells are contacted ex vivo with replication incompetentrecombinant retroviral particles to genetically modify T cells and/or NKcells to illicit a targeted immune response in the subject whenreintroduced into the subject. During the period of contact, thereplication incompetent recombinant retroviral particles identify andbind to T cells and/or NK cells at which point the retroviral and hostcell membranes start to fuse. Then, through the process of transduction,genetic material from the replication incompetent recombinant retroviralparticles enters the T cells and/or NK cells and is incorporated intothe host cell DNA. Methods of lentiviral transduction are known.Exemplary methods are described in, e.g., Wang et al. (2012) J.Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644;Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri etal. (2003) Blood. 102(2): 497-505.

Many of the methods provided herein include transduction of T cellsand/or NK cells. Methods are known in the art for transducing T cellsand/or NK cells ex vivo with replication incompetent recombinantretroviral particles, such as replication incompetent recombinantlentiviral particles. Methods provided herein, in illustrativeembodiments, do not require ex vivo stimulation or activation. Thus,this common step in prior methods can be avoided in the present method,although ex vivo stimulatory molecule(s) such as anti-CD3 and/oranti-CD28 beads, can be present during the transduction. However, withillustrative methods provided herein, ex vivo stimulation is notrequired. In certain exemplary methods, between 3 and 10 multiplicity ofinfection (MOI), and in some embodiments, between 5 and 10 MOI units ofreplication incompetent recombinant retroviral particles, for examplelentivirus, can be used.

The transduction reaction can be carried out in a closed system, such asa Sepax system, as discussed herein, wherein the transduction reactioncan be carried out in disposable bags loaded on the system. Blood cells,such as PBMCs, from the collected blood sample from the subject, can becontacted with replication incompetent recombinant retroviral particlesdisclosed herein, in a bag as soon as these blood cells are separated,isolated, and/or purified away from granulocytes, including neutrophils,which are typically not present during the contacting step (i.e. thetransduction reaction).

The replication incompetent recombinant retroviral particles can beintroduced into the bag that contains the isolated PBMCs, therebycontacting the PBMCs. The time from blood collection from the subject tothe time when blood cells, such as PBMCs are added to the transductionreaction bag, can be between 30 minutes and 4 hours, between 30 minutesand 2 hours, or around 1 hour, in some examples. Additives such asmedia, human serum albumin, human AB+ serum, and/or serum derived fromthe subject can be added to the transduction reaction mixture. Media istypically present, such as those known in the art for ex vivo processes(as non-limiting examples, X-VIVO 15 (Lonza) or CTS media (Thermo FisherScientific). Supportive cytokines can be added to the transductionreaction mixture, such as IL2, IL7, or IL15, or those found in HSA.

The transduction reaction mixture can be incubated at between 23 and 39°C., and in some illustrative embodiments at 37° C. In certainembodiments, the transduction reaction can be carried out at 37-39° C.for faster fusion/transduction. dGTP can be added to the transductionreaction. The transduction reaction mixture can be incubated for 1 to 12hours, and in some embodiments, 6 to 12 hrs before a wash is performedregardless of whether such cells will be studied in vitro, ex vivo orintroduced into a subject. Accordingly, in certain embodiments wherecells are infused into a subject, after transduction, before thetransduced T cells and/or NK cells are infused back into the subject,the cells are washed to remove the transduction reaction mixture beforebeing infused back into the subject. For example, the system, such as aSepax instrument, can be used to wash cells, for example with 10-50 mlof wash solution, before the transduced cells are infused back into thesubject. In some embodiments, neutrophils are removed before PBMCsand/or T cells and/or NK cells are processed, contacted with replicationincompetent recombinant retroviral particles, transduced, ortransfected.

In an illustrative embodiment for performing adoptive cell therapy,blood is collected from a subject into a blood bag and the blood bag isattached to a cell processing system such as a Sepax cell processingsystem. PBMCs isolated using the cell processing system are collectedinto a bag, contacted with the replication incompetent recombinantretroviral particles in conditions sufficient to transduce T cellsand/or NK cells, and incubated. After incubation, the bag containing themixture of PBMCs and replication incompetent recombinant retroviralparticles is attached to a cell processing system and the PBMCs arewashed. The washed PBMCs are collected into a bag and reinfused into thesubject. In some embodiments, the entire method, from collecting bloodto reinfusing transduced T and/or NK cells, is performed within 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, or 24 hours. In illustrativeembodiments, the entire method is performed within 12 hours.

In some embodiments, the target cells for the replication incompetentrecombinant retroviral particles are PBLs. In some embodiments, thetarget cells are T cells and/or NK cells. In some embodiments, the Tcells are helper T cells and/or killer T cells.

In some embodiments, the replication incompetent recombinant retroviralparticles provided herein have pseudotyping elements on their surfacethat are capable of binding to T cells and/or NK cells and facilitatingmembrane fusion of the replication incompetent recombinant retroviralparticles thereto. In other embodiments, the replication incompetentrecombinant retroviral particles have activation elements on theirsurface that are capable of binding to resting T cells and/or NK cells.In still other embodiments, the replication incompetent recombinantretroviral particles have membrane-bound cytokines on their surface. Insome embodiments, the replication incompetent recombinant retroviralparticles include a polynucleotide having one or more transcriptionalunits encoding one or more engineered signaling polypeptides, one ormore of which includes one or more lymphoproliferative elements. Inother embodiments, when two signaling polypeptides are utilized, oneincludes at least one lymphoproliferative element and the other istypically a chimeric antigen receptor (CAR) that includes anantigen-specific targeting region (ASTR), a transmembrane domain, and anintracellular activating domain. As indicated herein, an activationelement(s) that is typically associated with the surface of areplication incompetent recombinant retroviral particle provided herein,is capable of, and as a resulting of contacting resting T cells and/orNK cells for a sufficient period of time and under appropriateconditions, activates resting T cells and/or NK cells. It will beunderstood that such activation occurs over time during a contactingstep of methods herein. Furthermore, it will be understood that in someembodiments where a pseudotyping element is found on the surface of areplication incompetent recombinant retroviral particle, that binds a Tcell and/or an NK cell, in methods herein, activation can be induced bybinding of the pseudotyping element. An activation element is optionalin those embodiments.

Further details regarding a pseudotyping element, an activation element,a membrane-bound cytokine, an engineered signaling polypeptide, alymphoproliferative element, and a CAR are provided in other sectionsherein.

In some embodiments of the methods and compositions disclosed herein,between 5% and 90% of the total lymphocytes collected from the blood aretransduced. In some embodiments, the percent of lymphocytes that aretransduced is between 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60%on the low end of the range, and 50, 55, 60, 65, 70, 75, 80, 85, and 90%on the high end of the range. In some embodiments, the percent oflymphocytes that are transduced is at least 5%, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, or at least 60%.

In some embodiments of the methods and compositions disclosed herein,the genetically modified T cells and/or NK cells are introduced back,reintroduced, or reinfused into the subject without additional ex vivomanipulation, such as stimulation and/or activation of T cells and/orNKs. In the prior art methods, ex vivo manipulation is used forstimulation/activation of T cells and/or NK cells and for expansion ofgenetically modified T cells and/or NK cells prior to introducing thegenetically modified T cells and/or NK cells into the subject. In priorart methods, this generally takes days or weeks and requires a subjectto return to a clinic for a blood infusion days or weeks after aninitial blood draw. In some embodiments of the methods and compositionsdisclosed herein, T cells and/or NK cells are not stimulated ex vivo byexposure to anti-CD3/anti-CD28 solid supports such as, for example,beads coated with anti-CD3/anti-CD28, prior to contacting the T cellsand/or NK cells with the replication incompetent recombinant retroviralparticles. As such provided herein is an ex vivo propagation-freemethod. In other embodiments, genetically modified T cells and/or NKcells are not expanded ex vivo, or only expanded for a small number ofcell divisions (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 rounds of celldivision), but are rather expanded, or predominantly expanded, in vivo,i.e. within the subject. In some embodiments, no additional media isadded to allow for further expansion of the cells. In some embodiments,no cell manufacturing of the PBLs occurs while the PBLs are contactedwith the replication incompetent recombinant retroviral particles. Inillustrative embodiments, no cell manufacturing of the PBLs occurs whilethe PBLs are ex vivo. In previous methods of adoptive cell therapy,subjects were lymphodepleted prior to reinfusion with geneticallymodified T cells and or NK cells. In some embodiments, patients orsubjects are not lymphodepleted prior to blood being withdrawn. In someembodiments, patients or subjects are not lymphodepleted prior toreinfusion with genetically modified T cells and or NK cells.

In any of the embodiments disclosed herein, the number of T cells and/orNK cells to be reinfused into a subject can be between 1×10³, 2.5×10³,5×10³, 1×10⁴, 2.5×10⁴, 5×10⁴, 1×10⁵, 2.5×10⁵, 5×10⁵, 1×10⁶, 2.5×10⁶,5×10⁶, and 1×10⁷ cells/kg on the low end of the range and 5×10⁴, 1×10⁵,2.5×10⁵, 5×10⁵, 1×10⁶, 2.5×10⁶, 5×10⁶, 1×10⁷, 2.5×10⁷, 5×10⁷, and 1×10⁸cells/kg on the high end of the range. In illustrative embodiments, thenumber of T cells and/or NK cells to be reinfused into a subject can bebetween 1×10⁴, 2.5×10⁴, 5×10⁴, and 1×10⁵ cells/kg on the low end of therange and 2.5×10⁴, 5×10⁴, 1×10⁵, 2.5×10⁵, 5×10⁵, and 1×10⁶ cells/kg onthe high end of the range. In some embodiments, the number of PBLs to bereinfused into a subject can be fewer than 5×10⁵, 1×10⁶, 2.5×10⁶, 5×10⁶,1×10⁷, 2.5×10⁷, 5×10⁷, and 1×10⁸ cells and the low end of the range and2.5×10⁶, 5×10⁶, 1×10⁷, 2.5×10⁷, 5×10⁷, 1×10⁸, 2.5×10⁸, 5×10⁸, and 1×10⁹cells on the high end of the range. In some embodiments, the number of Tcells and/or NK cells available for reinfusion into a 70 kg subject orpatient is between 7×10⁵ and 2.5×10⁸ cells. In other embodiments, thenumber of T cells and/or NK cells available for transduction isapproximately 7×10⁶ plus or minus 10%.

In the methods disclosed herein, the entire adoptive cell therapyprocedure, from withdrawing blood to the reinfusion of geneticallymodified T cells and/or NK cells, can advantageously be performed in ashorter time than previous methods. In some embodiments, the entireadoptive cell therapy procedure can be performed in less than 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, or 24 hours. In illustrativeembodiments, the entire adoptive cell therapy procedure can be performedin less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours. In someembodiments, the entire adoptive cell therapy procedure can be performedin between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 15 hours on the lowend of the range and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, or24 hours on the high end of the range.

In some embodiments herein, a closed system is used to process PBMCs,for example in methods that include genetically modifying PBMCs, NKcells and in illustrative embodiments T cells, for example bytransducing the PBMCs or subset(s) thereof. Such methods can be used togenetically modify lymphocytes to be used in scientific research,commercial production, or therapeutic methods. For example, such methodscan include transferring peripheral blood mononuclear cells (PBMCs)including NK cells, T cells, or both, and in some embodiments resting Tcells, and/or resting NK cells, from a vessel into a transductionreaction mixture within a closed system, which thus is withoutenvironmental exposure. The vessel, for example, can be a tube, bag,syringe, or other container. In some embodiments, the vessel is a vesselthat is used in a research facility. In some embodiments, the vessel isa vessel used in commercial production. In other embodiments, the vesselcan be a collection vessel used in a blood collection process. Methodsfor genetically modifying herein, typically involve a contacting stepwherein lymphocytes are contacted with a replication incompetentrecombinant retroviral particle. The contacting in some embodiments, canbe performed in the vessel, for example, within a blood bag. In otherembodiments, PBMCs or a subfraction thereof, can be transferred from thevessel to another vessel (for example from a first vessel to a secondvessel) within the closed system for the contacting. The second vesselcan be a cell processing compartment of a closed device, such as a G-Rexdevice. In some embodiments, after the contacting the geneticallymodified (e.g. transduced) cells can be transferred to a differentvessel within the closed system (i.e. without exposure to theenvironment). Either before or after this transfer the cells aretypically washed within the closed system to remove substantially all orall of the retroviral particles. In some embodiments, the processdisclosed in this paragraph from transfer of the PBMCs or a fractionthereof, into the vessel in which the contacting (e.g. transduction)will occur through washing the cells after the contacting, is performedwithin 12 hours. In some embodiments it is performed for between 1, 2,3, or 4 hours on the low end of the range, and 4, 8, 10, or 12 hours onthe high end of the range.

In some embodiments provided herein, the steps of withdrawing a bloodsample from a subject, contacting T cells and/or NK cells withreplication incompetent recombinant retroviral particles, and/orintroducing genetically modified T cells and/or NK cells into thesubject, occur in a closed system. A closed system is a culture processthat is generally closed or fully closed to contamination. As such, sucha system or process does not expose cells to an environment. Anadvantage of the present invention, is that provided herein are methodsfor performing CAR therapy in a closed system. One of the greatest risksto safety and regulatory control in the cell processing procedure is therisk of contamination through frequent exposure to the environment as isfound in traditional open cell culture systems. To mitigate this risk,particularly in the absence of antibiotics, some commercial processeshave been developed that focus on the use of disposable (single-use)equipment. However even with their use under aseptic conditions, thereis always a risk of contamination from the opening of flasks to sampleor add additional growth media. To overcome this problem, providedherein is a closed-system process, a process that is designed and can beoperated such that the product is not exposed to the outsideenvironment. This is important because the outside environment istypically not sterile. Material transfer occurs via sterile connectionsor tube welding. Air for gas exchange occurs via a gas permeablemembrane or like other additions, via 0.2 μm filter to preventenvironmental exposure.

In some embodiments, the closed system includes an ex vivo circulatingsystem connected to the in vivo circulatory system of the subject suchthat blood is drawn and then circulated to the ex vivo circulatorysystem before being introduced back into the subject. In someembodiments, the ex vivo circulatory system includes a system orapparatus for isolating PBLs and/or a system or apparatus for isolatingT cells and/or NK cells, in combination with the system or apparatus forexposing the cells to the replication incompetent recombinant retroviralparticles. In some embodiments, the closed system does not allow the Tcells and/or NK cells to be exposed to air.

Such closed system methods can be performed with commercially availabledevices. For example, the method can be carried out in devices adaptedfor closed system T cell production. Such devices include a G-Rex™, aWAVE Bioreactor™, an OriGen PermaLife™ bags, and a VueLife® bags.

In some embodiments of the methods and compositions disclosed herein,genetically modified T cells and/or NK cells within a subject areexposed to a compound that binds to an in vivo control element presenttherein, in which the control element is a part of the genetic materialintroduced by the replication incompetent recombinant retroviralparticles. In some embodiments, the control element can be a riboswitchand the compound can bind the aptamer domain of the riboswitch. In someembodiments, the control element can be a molecular chaperone. In any ofthe embodiments disclosed herein, the compound can be a nucleosideanalogue. In some embodiments, the nucleoside analogue can be anucleoside analogue antiviral drug, wherein an antiviral drug is acompound approved by the Food and Drug Administration for antiviraltreatment or a compound in an antiviral clinical trial in the UnitedStates. In illustrative embodiments, the compound can be acyclovir orpenciclovir. In some embodiments, the compound can be famciclovir, theoral prodrug of penciclovir, or valaciclovir, the oral prodrug ofacyclovir. Binding of the compound to the control element affectsexpression of the introduced genetic material and hence, propagation ofgenetically modified T cells and/or NK cells.

In some embodiments, the nucleoside analogue antiviral drug or prodrug,for example acyclovir, valaciclovir, penciclovir or famciclovir, isadministered to the subject prior to, concurrent with, and/or followingPBLs being isolated from the blood of the subject and before T cellsand/or NK cells are contacted with replication incompetent recombinantretroviral particles. In some embodiments, the nucleoside analogueantiviral drug or prodrug is administered to the subject for between 5,10, 15, 30, and 60 minutes on the low end of the range and 1.5, 2, 3, 4,5, 6, 8, 12, or 24 hours on the high end of the range prior to PBLsbeing isolated from the blood or prior to T cells and/or NK cells beingcontacted with replication incompetent recombinant retroviral particles.In other embodiments, the nucleoside analogue antiviral drug or prodrugis administered to the subject for between 1.5, 2, 3, 4, 5, 6, 8, 12, or24 hours on the low end of the range and ½, 1, 2, 3, 4, 5, 6, 7, 10, 14,21, or 28 days on the high end of the range after PBLs are isolated fromthe blood and T cells and/or NK cells are contacted with replicationincompetent recombinant retroviral particles in methods provided herein.In some embodiments, the nucleoside analogue antiviral drug or prodrugis administered to the subject for at least 1.5, 2, 3, 4, 5, 6, 8, 12,or 24 hours, or at least 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 days afterPBLs are isolated from the blood and T cells and/or NK cells arecontacted with replication incompetent recombinant retroviral particlesin methods provided herein. In some embodiments, the nucleoside analogueantiviral drug or prodrug is administered to the subject for at least 1,2, 3, 4, 5, 7, 10, 14, 21, 28, 30, 60, 90, or 120 days or 5, 6, 9, 12,24, 36, 48, 60, 72, 84, 96, 120 months or indefinitely after the PBLshave been reinfused into the subject. In any of the embodimentsdisclosed herein, the nucleoside analogue antiviral drug or prodrug canbe administered before and/or during the reinfusion of the PBLs and/orafter the PBLs have been reinfused.

In some embodiments, the compound that binds to the control element isadministered once, twice, three times, or four times daily to thesubject. In some embodiments, daily doses of the compound are providedfor 1 week, 2 weeks, 4 weeks, 3 months, 6 months, 1 year, until asubject is disease free, such as cancer free, or indefinitely. The drug,in illustrative embodiments is a nucleoside analogue antiviral drug thatbinds to a nucleoside analog, such as a riboswitch, as disclosed infurther detail herein.

Methods are known in the art for delivering drugs, whether smallmolecules or biologics, and can be used in methods provided herein. Anysuch methods can be used to deliver drugs or candidate compounds orantibodies for use in methods of the present invention. For example,common routes of administration include non-invasive peroral (throughthe mouth), topical (skin), transmucosal (nasal, buccal/sublingual,vaginal, ocular and rectal) and inhalation routes. Many protein andpeptide drugs, such as monoclonal antibodies, have to be delivered byinjection or a nanoneedle array. For example, many immunizations arebased on the delivery of protein drugs and are often done by injection.

Engineered Signaling Polypeptide(s)

In some embodiments, the replication incompetent recombinant retroviralparticles used to contact T cells and/or NK cells have a polynucleotidehaving one or more transcriptional units that encode one or moreengineered signaling polypeptides. In some embodiments, an engineeredsignaling polypeptide includes any combination of an extracellulardomain (e.g. an antigen-specific targeting region or ASTR), an optionala stalk and a transmembrane domain, combined with one or moreintracellular activating domains, one or more modulatory domains (suchas a co-stimulatory domain), and one or more T cell survival motifs. Inillustrative embodiments, at least one, two, or all of the engineeredsignaling polypeptides is a chimeric antigen receptor (CAR) or alymphoproliferative element including a chimeric lymphoproliferativeelement (CLE). In some embodiments, when two signaling polypeptides areutilized, one encodes a lymphoproliferative element and the otherencodes a chimeric antigen receptor (CAR) that includes anantigen-specific targeting region (ASTR), a transmembrane domain, and anintracellular activating domain. One of ordinary skill in the art wouldbe able to configure the system to put the lymphoproliferative elementand the CAR on distinct polynucleotides with similar or dissimilarcontrol elements for the methods and compositions disclosed herein. Askilled artisan will recognize that such engineered polypeptides canalso be referred to as recombinant polypeptides.

Extracellular Domain

In some embodiments, an engineered signaling polypeptide includes anextracellular domain that is a member of a specific binding pair. Forexample, in some embodiments, the extracellular domain can be theextracellular domain of a cytokine receptor, or a mutant thereof, or ahormone receptor, or a mutant thereof. Such mutant extracellular domainsin some embodiments have been reported to be constitutively active whenexpressed at least in some cell types. In illustrative embodiments, suchextracellular and transmembrane domains do not include a ligand bindingregion. It is believed that such domains do not bind a ligand whenpresent in an engineered signaling polypeptide and expressed in B cells,T cells, and/or NK cells. Mutations in such receptor mutants can occurin the extracellular juxtamembrane region. Not to be limited by theory,a mutation in at least some extracellular domains (and someextracellular-transmembrane domains) of engineered signalingpolypeptides provided herein, are responsible for signaling of theengineered signaling polypeptide in the absence of ligand, by bringingactivating chains together that are not normally together. Furtherembodiments regarding extracellular domains that comprise mutations inextracellular domains can be found, for example, in theLymphoproliferative Element section herein.

In certain illustrative embodiments, the extracellular domain comprisesa dimerizing motif. In an illustrative embodiment the dimerizing motifcomprises a leucine zipper. In some embodiments, the leucine zipper isfrom a jun polypeptide, for example c-jun. Further embodiments regardingextracellular domains that comprise a dimerizing motif can be found, forexample, in the Lymphoproliferative Element section herein.

In certain embodiments, the extracellular domain is an antigen-specifictargeting region (ASTR), sometimes called an antigen binding domainherein. Specific binding pairs include, but are not limited to,antigen-antibody binding pairs; ligand-receptor binding pairs; and thelike. Thus, a member of a specific binding pair suitable for use in anengineered signaling polypeptide of the present disclosure includes anASTR that is an antibody, an antigen, a ligand, a receptor bindingdomain of a ligand, a receptor, a ligand binding domain of a receptor,and an affibody.

An ASTR suitable for use in an engineered signaling polypeptide of thepresent disclosure can be any antigen-binding polypeptide. In certainembodiments, the ASTR is an antibody such as a full-length antibody, asingle-chain antibody, an Fab fragment, an Fab′ fragment, an (Fab′)2fragment, an Fv fragment, and a divalent single-chain antibody or adiabody.

In some embodiments, the ASTR is a single chain Fv (scFv). In someembodiments, the heavy chain is positioned N-terminal of the light chainin the engineered signaling polypeptide. In other embodiments, the lightchain is positioned N-terminal of the heavy chain in the engineeredsignaling polypeptide. In any of the disclosed embodiments, the heavyand light chains can be separated by a linker as discussed in moredetail herein. In any of the disclosed embodiments, the heavy or lightchain can be at the N-terminus of the engineered signaling polypeptideand is typically C-terminal of another domain, such as a signal sequenceor peptide.

Other antibody-based recognition domains (cAb VHH (camelid antibodyvariable domains) and humanized versions, IgNAR VH (shark antibodyvariable domains) and humanized versions, sdAb VH (single domainantibody variable domains) and “camelized” antibody variable domains aresuitable for use with the engineered signaling polypeptides and methodsusing the engineered signaling polypeptides of the present disclosure.In some instances, T cell receptor (TCR) based recognition domains suchas single chain TCR (scTv, single chain two-domain TCR containing VαVβ)are also suitable for use.

In some embodiments, the ASTR can be multispecific, e.g. bispecificantibodies. Multispecific antibodies have binding specificities for atleast two different sites. In certain embodiments, one of the bindingspecificities is for one target antigen and the other is for anothertarget antigen. In certain embodiments, bispecific antibodies may bindto two different epitopes of ta target antigen. Bispecific antibodiesmay also be used to localize cytotoxic agents to cells which express atarget antigen. Bispecific antibodies can be prepared as full lengthantibodies or antibody fragments.

An ASTR suitable for use in an engineered signaling polypeptide of thepresent disclosure can have a variety of antigen-binding specificities.In some cases, the antigen-binding domain is specific for an epitopepresent in an antigen that is expressed by (synthesized by) a targetcell. In one example, the target cell is a cancer cell associatedantigen. The cancer cell associated antigen can be an antigen associatedwith, e.g., a breast cancer cell, a B cell lymphoma, a Hodgkin lymphomacell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma, alung cancer cell (e.g., a small cell lung cancer cell), a non-HodgkinB-cell lymphoma (B-NHL) cell, an ovarian cancer cell, a prostate cancercell, a mesothelioma cell, a lung cancer cell (e.g., a small cell lungcancer cell), a melanoma cell, a chronic lymphocytic leukemia cell, anacute lymphocytic leukemia cell, a neuroblastoma cell, a glioma, aglioblastoma, a medulloblastoma, a colorectal cancer cell, etc. A cancercell associated antigen may also be expressed by a non-cancerous cell.

Non-limiting examples of antigens to which an ASTR of an engineeredsignaling polypeptide can bind include, e.g., CD19, CD20, CD38, CD30,ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA),epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelialgrowth factor receptor-2 (VEGFR2), high molecular weight-melanomaassociated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, Ax1, Ror2, andthe like.

In some cases, a member of a specific binding pair suitable for use inan engineered signaling polypeptide is an ASTR that is a ligand for areceptor. Ligands include, but are not limited to, hormones (e.g.erythropoietin, growth hormone, leptin, etc.); cytokines (e.g.,interferons, interleukins, certain hormones, etc.); growth factors(e.g., heregulin; vascular endothelial growth factor (VEGF); and thelike); an integrin-binding peptide (e.g., a peptide comprising thesequence Arg-Gly-Asp); and the like.

Where the member of a specific binding pair in an engineered signalingpolypeptide is a ligand, the engineered signaling polypeptide can beactivated in the presence of a second member of the specific bindingpair, where the second member of the specific binding pair is a receptorfor the ligand. For example, where the ligand is VEGF, the second memberof the specific binding pair can be a VEGF receptor, including a solubleVEGF receptor.

As noted above, in some cases, the member of a specific binding pairthat is included in an engineered signaling polypeptide is an ASTR thatis a receptor, e.g., a receptor for a ligand, a co-receptor, etc. Thereceptor can be a ligand-binding fragment of a receptor. Suitablereceptors include, but are not limited to, a growth factor receptor(e.g., a VEGF receptor); a killer cell lectin-like receptor subfamily K,member 1 (NKG2D) polypeptide (receptor for MICA, MICB, and ULB6); acytokine receptor (e.g., an IL-13 receptor; an IL-2 receptor; etc.);CD27; a natural cytotoxicity receptor (NCR) (e.g., NKP30 (NCR3/CD337)polypeptide (receptor for HLA-B-associated transcript 3 (BAT3) andB7-H6); etc.); etc.

In certain embodiments of any of the aspects provided herein thatinclude an ASTR, the ASTR can be directed to an intermediate proteinthat links the ASTR with a target molecule expressed on a target cell.The intermediate protein may be endogenously expressed or introducedexogenously and may be natural, engineered, or chemically modified. Incertain embodiments the ASTR can be an anti-tag ASTR such that at leastone tagged intermediate, typically an antibody-tag conjugate, isincluded between a tag recognized by the ASTR and a target molecule,typically a protein target, expressed on a target cell. Accordingly, insuch embodiments, the ASTR binds a tag and the tag is conjugated to anantibody directed against an antigen on a target cell, such as a cancercell. Non-limiting examples of tags include fluorescein isothiocyanate(FITC), streptavidin, biotin, histidine, dinitrophenol, peridininchlorophyll protein complex, green fluorescent protein, phycoerythrin(PE), horse radish peroxidase, palmitoylation, nitrosylation, alkalaninephosphatase, glucose oxidase, and maltose binding protein. As such, theASTR comprises a molecule that binds the tag.

Stalk

In some embodiments, the engineered signaling polypeptide includes astalk which is located in the portion of the engineered signalingpolypeptide lying outside the cell and interposed between the ASTR andthe transmembrane domain. In some cases, the stalk has at least 85, 90,95, 96, 97, 98, 99, or 100% identity to a wild-type CD8 stalk region(TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG AVHTRGLDFA (SEQ ID NO:79), has atleast 85, 90, 95, 96, 97, 98, 99, or 100% identity to a wild-type CD28stalk region (FCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ IDNO:80)), or has at least 85, 90, 95, 96, 97, 98, 99, or 100% identity toa wild-type immunoglobulin heavy chain stalk region. In an engineeredsignaling polypeptide, the stalk employed allows the antigen-specifictargeting region, and typically the entire engineered signalingpolypeptide, to retain increased binding to a target antigen.

The stalk region can have a length of from about 4 amino acids to about50 amino acids, e.g., from about 4 aa to about 10 aa, from about 10 aato about 15 aa, from about 15 aa to about 20 aa, from about 20 aa toabout 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about40 aa, or from about 40 aa to about 50 aa.

In some cases, the stalk of an engineered signaling polypeptide includesat least one cysteine. For example, in some cases, the stalk can includethe sequence Cys-Pro-Pro-Cys (SEQ ID NO:62). If present, a cysteine inthe stalk of a first engineered signaling polypeptide can be availableto form a disulfide bond with a stalk in a second engineered signalingpolypeptide.

Stalks can include immunoglobulin hinge region amino acid sequences thatare known in the art; see, e.g., Tan et al. (1990) Proc. Natl. Acad.Sci. USA 87:162; and Huck et al. (1986) Nucl. Acids Res. 14:1779. Asnon-limiting examples, an immunoglobulin hinge region can include adomain with at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or100% sequence identity to a stretch of at least 10, 15, 20, or all ofthe amino acids of any of the following amino acid sequences: DKTHT (SEQID NO:63); CPPC (SEQ ID NO:62); CPEPKSCDTPPPCPR (SEQ ID NO:64) (see,e.g., Glaser et al. (2005) J. Biol. Chem. 280:41494); ELKTPLGDTTHT (SEQID NO:65); KSCDKTHTCP (SEQ ID NO:66); KCCVDCP (SEQ ID NO:67); KYGPPCP(SEQ ID NO:68); EPKSCDKTHTCPPCP (SEQ ID NO:69) (human IgG1 hinge);ERKCCVECPPCP (SEQ ID NO:70) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQID NO:71) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO:72) (human IgG4hinge); and the like. The stalk can include a hinge region with an aminoacid sequence of a human IgG1, IgG2, IgG3, or IgG4, hinge region. Thestalk can include one or more amino acid substitutions and/or insertionsand/or deletions compared to a wild-type (naturally-occurring) hingeregion. For example, His229 of human IgG 1 hinge can be substituted withTyr, so that the stalk includes the sequence EPKSCDKTYTCPPCP (see, e.g.,Yan et al. (2012) J. Biol. Chem. 287:5891). The stalk can include anamino acid sequence derived from human CD8; e.g., the stalk can includethe amino acid sequence:

-   -   TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:73), or        a variant thereof.

Transmembrane Domain

An engineered signaling polypeptide of the present disclosure caninclude transmembrane domains for insertion into a eukaryotic cellmembrane. The transmembrane domain can be interposed between the ASTRand the co-stimulatory domain. The transmembrane domain can beinterposed between the stalk and the co-stimulatory domain, such thatthe chimeric antigen receptor includes, in order from the amino terminus(N-terminus) to the carboxyl terminus (C-terminus): an ASTR; a stalk; atransmembrane domain; and an activating domain.

Any transmembrane (TM) domain that provides for insertion of apolypeptide into the cell membrane of a eukaryotic (e.g., mammalian)cell is suitable for use in aspects and embodiments disclosed herein.Non-limiting examples of TM domains suitable for any of the aspects orembodiments provided herein, include a domain with at least 50, 60, 70,75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity to astretch of at least 10, 15, 20, or all of the amino acids of any of thefollowing TM domains or combined stalk and TM domains: a) CD8 alpha TM(SEQ ID NO:46); b) CD8 beta TM (SEQ ID NO:47); c) CD4 stalk (SEQ IDNO:48); d) CD3Z TM (SEQ ID NO:49); e) CD28 TM (SEQ ID NO:50); f) CD134(OX40) TM: (SEQ ID NO:51); g) CD7 TM (SEQ ID NO:52); h) CD8 stalk and TM(SEQ ID NO:75); and i) CD28 stalk and TM (SEQ ID NO:76).

As non-limiting examples, a transmembrane domain of an aspect of theinvention can have at least 80, 90, or 95% sequence identity to the SEQID NO:46 transmembrane domain, the CD8 beta transmembrane domain, theCD4 transmembrane domain, the CD3 zeta transmembrane domain, the CD28transmembrane domain, the CD134 transmembrane domain, or the CD7transmembrane domain.

Intracellular Activating Domain

Intracellular activating domains suitable for use in an engineeredsignaling polypeptide of the present disclosure when activated,typically induce the production of one or more cytokines; increased celldeath; and/or increased proliferation of CD8⁺ T cells, CD4⁺ T cells, NKTcells, γδ T cells, and/or neutrophils. Activating domains can also bereferred to as activation domains herein. Activating domains can be usedin CARs or in lymphoproliferative elements provided herein.

In some embodiments, the intracellular activating domain includes atleast one (e.g., one, two, three, four, five, six, etc.) ITAM motifs asdescribed below. In some embodiments, an intracellular activating domainof an aspect of the invention can have at least 80%, 90%, or 95%sequence identity to the CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12,FCERIG, FCGR2A, FCGR2C.

DAP10/CD28, or ZAP70 domains as described below.

Intracellular activating domains suitable for use in an engineeredsignaling polypeptide of the present disclosure include immunoreceptortyrosine-based activation motif (ITAM)-containing intracellularsignaling polypeptides. An ITAM motif is YX₁X₂L/I, where X₁ and X₂ areindependently any amino acid. In some cases, the intracellularactivating domain of an engineered signaling polypeptide includes 1, 2,3, 4, or 5 ITAM motifs. In some cases, an ITAM motif is repeated twicein an intracellular activating domain, where the first and secondinstances of the ITAM motif are separated from one another by 6 to 8amino acids, e.g., (YX₁X₂L/I)(X₃)_(n)(YX₁X₂L/I), where n is an integerfrom 6 to 8, and each of the 6-8 X₃ can be any amino acid. In somecases, the intracellular activating domain of an engineered signalingpolypeptide includes 3 ITAM motifs.

A suitable intracellular activating domain can be an ITAMmotif-containing portion that is derived from a polypeptide thatcontains an ITAM motif. For example, a suitable intracellular activatingdomain can be an ITAM motif-containing domain from any ITAMmotif-containing protein. Thus, a suitable intracellular activatingdomain need not contain the entire sequence of the entire protein fromwhich it is derived. Examples of suitable ITAM motif-containingpolypeptides include, but are not limited to: CD3Z (CD3 zeta); CD3D (CD3delta); CD3E (CD3 epsilon); CD3G (CD3 gamma); CD79A (antigen receptorcomplex-associated protein alpha chain); DAP12; and FCERIG (Fc epsilonreceptor I gamma chain).

In some cases, the intracellular activating domain is derived from Tcell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cellreceptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.).For example, a suitable intracellular activating domain can include adomain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20,or all amino acids in the following sequences or to a contiguous stretchof from about 100 amino acids to about 110 amino acids (aa), from about110 aa to about 115 aa, from about 115 aa to about 120 aa, from about120 aa to about 130 aa, from about 130 aa to about 140 aa, from about140 aa to about 150 aa, or from about 150 aa to about 160 aa, of eitherof the following amino acid sequences (2 isoforms):

-   -   MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQ        GQNQL[YNELNLGRREEYDVL]DKRRGRDPEMGGKPRRKNPQEGL[YNELQKDKMAEAYSEI]G        MKGERRRGKGHDGL[YQGLSTATKDTYDAL]HMQALPPR (SEQ ID NO:11) or    -   MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQ        GQNQL[YNELNLGRREEYDVL]DKRRGRDPEMGGKPQRRKNPQEGL[YNELQKDKMAEAYSEI]GMKGERRRGKGHDGL[YQGLSTATKDTYDAL]HMQALPPR        (SEQ ID NO:12), where the ITAM motifs are set out with brackets.

Likewise, a suitable intracellular activating domain polypeptide caninclude an

ITAM motif-containing a portion of the full length CD3 zeta amino acidsequence. Thus, a suitable intracellular activating domain can include adomain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20,or all amino acids in the following sequences or to a contiguous stretchof from about 100 amino acids to about 110 amino acids (aa), from about110 aa to about 115 aa, from about 115 aa to about 120 aa, from about120 aa to about 130 aa, from about 130 aa to about 140 aa, from about140 aa to about 150 aa, or from about 150 aa to about 160 aa, of eitherof the following amino acid sequences:

-   -   RVKFSRSADAPAYQQGQNQL[YNELNLGRREEYDVL]DKRRGRDPEMGGKPRRKNPQEGL[YNE        LQKDKMAEAYSEI]GMKGERRRGKGHDGL[YQGLSTATKDTYDAL]HMQALPPR (SEQ ID        NO:13);    -   RVKFSRSADAPAYQQGQNQL[YNELNLGRREEYDVL]DKRRGRDPEMGGKPQRRKNPQEGL[YN        ELQKDKMAEAYSEI]GMKGERRRGKGHDGL[YQGLSTATKDTYDAL]HMQALPPR (SEQ ID        NO:81); NQL[YNELNLGRREEYDVL]DKR SEQ ID NO:14);        EGL[YNELQKDKMAEAYSEI]GMK (SEQ ID NO:15); or        DGL[YQGLSTATKDTYDAL]HMQ (SEQ ID NO:16), where the ITAM motifs        are set out in brackets.

In some cases, the intracellular activating domain is derived from Tcell surface glycoprotein CD3 delta chain (also known as CD3D;CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen,delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3delta chain; T cell surface glycoprotein CD3 delta chain; etc.). Thus, asuitable intracellular activating domain can include a domain with atleast 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to a stretch of at least 10, 15, 20, or all aminoacids in the following sequences or to a contiguous stretch of fromabout 100 amino acids to about 110 amino acids (aa), from about 110 aato about 115 aa, from about 115 aa to about 120 aa, from about 120 aa toabout 130 aa, from about 130 aa to about 140 aa, from about 140 aa toabout 150 aa, or from about 150 aa to about 160 aa, of either

of the following amino acid sequences:

-   -   MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDP        RGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGR        LSGAADTQALLRNDQV[YQPLRDRDDAQYSHL]GGNWARNK (SEQ ID NO:17) or    -   MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDP        RGIYRCNGTDIYKDKESTVQVHYRTADTQALLRNDQV[YQPLRDRDDAQYSHL]GGNWARNK        (SEQ ID NO:18), where the ITAM motifs are set out in brackets.

Likewise, a suitable intracellular activating domain polypeptide cancomprise an ITAM motif-containing portion of the full length CD3 deltaamino acid sequence. Thus, a suitable intracellular activating domaincan include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of atleast 10, 15, 20, or all amino acids in the following sequence:DQV[YQPLRDRDDAQYSHL]GGN (SEQ ID NO:19), where the ITAM motifs are setout in brackets.

In some cases, the intracellular activating domain is derived from Tcell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cellsurface antigen T3/Leu-4 epsilon chain, T cell surface glycoprotein CD3epsilon chain, AI504783, CD3, CD3epsilon, T3e, etc.). Thus, a suitableintracellular activating domain can include a domain with at least 50%,60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to a stretch of at least 10, 15, 20, or all amino acids in thefollowing sequences or to a contiguous stretch of from about 100 aminoacids to about 110 amino acids (aa), from about 110 aa to about 115 aa,from about 115 aa to about 120 aa, from about 120 aa to about 130 aa,from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, orfrom about 150 aa to about 160 aa, of the following amino acid sequence:

-   -   MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDK        NIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDMS        VATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPD[YEPIRK        GQRDLYSGL]NQRRI (SEQ ID NO:20), where the ITAM motifs are set        out in brackets.

Likewise, a suitable intracellular activating domain polypeptide cancomprise an ITAM motif-containing portion of the full length CD3 epsilonamino acid sequence. Thus, a suitable intracellular activating domaincan include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of atleast 10, 15, 20, or all amino acids in the following sequence:NPD[YEPIRKGQRDLYSGL]NQR (SEQ ID NO:21), where the ITAM motifs are setout in brackets.

In some cases, the intracellular activating domain is derived from Tcell surface glycoprotein CD3 gamma chain (also known as CD3G, T cellreceptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3complex), etc.). Thus, a suitable intracellular activating domain caninclude a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least10, 15, 20, or all amino acids in the following sequences or to acontiguous stretch of from about 100 amino acids to about 110 aminoacids (aa), from about 110 aa to about 115 aa, from about 115 aa toabout 120 aa, from about 120 aa to about 130 aa, from about 130 aa toabout 140 aa, from about 140 aa to about 150 aa, or from about 150 aa toabout 160 aa, of the following amino acid sequence:

-   -   MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGF        LTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFV        LAVGVYFIAGQDGVRQSRASDKQTLLPNDQL[YQPLKDREDDQYSHL]QGNQLRRN (SEQ ID        NO:22), where the ITAM motifs are set out in brackets.

Likewise, a suitable intracellular activating domain polypeptide cancomprise an ITAM motif-containing portion of the full length CD3 gammaamino acid sequence. Thus, a suitable intracellular activating domaincan include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of atleast 10, 15, 20, or all amino acids in the following sequence:DQL[YQPLKDREDDQYSHL]QGN (SEQ ID NO:23), where the ITAM motifs are setout in brackets.

In some cases, the intracellular activating domain is derived from CD79A(also known as B-cell antigen receptor complex-associated protein alphachain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membraneglycoprotein; Ig-alpha; membrane-bound immunoglobulin-associatedprotein; surface IgM-associated protein; etc.). Thus, a suitableintracellular activating domain can include a domain with at least 50%,60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to a stretch of at least 10, 15, 20, or all amino acids in thefollowing sequences or to a contiguous stretch of from about 100 aminoacids to about 110 amino acids (aa), from about 110 aa to about 115 aa,from about 115 aa to about 120 aa, from about 120 aa to about 130 aa,from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, orfrom about 150 aa to about 160 aa, of either of the following amino acidsequences:

-   -   MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSSNNAN        VTWWRVLHGNYTWPPEFLGPGEDPNGTLIIQNVNKSHGGIYVCRVQEGNESYQQSCGTYLRVR        QPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENL[YEGL        NLDDCSMYEDI]SRGLQGTYQDVGSLNIGDVQLEKP (SEQ ID NO:24) or    -   MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSSNNAN        VTWWRVLHGNYTWPPEFLGPGEDPNEPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRK        RWQNEKLGLDAGDEYEDENL[YEGLNLDDCSMYEDI]SRGLQGTYQDVGSLNIGDVQLEKP        (SEQ ID NO:25), where the ITAM motifs are set out in brackets.

Likewise, a suitable intracellular activating domain polypeptide cancomprise an ITAM motif-containing portion of the full length CD79A aminoacid sequence. Thus, a suitable intracellular activating domain caninclude a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least10, 15, 20, or all amino acids in the following sequence:ENL[YEGLNLDDCSMYEDI]SRG (SEQ ID NO:26), where the ITAM motifs are setout in brackets.

In some cases, the intracellular activating domain is derived from DAP12(also

known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP;PLOSL; DNAX-activation protein 12; KAR-associated protein; TYRO proteintyrosine kinase-

binding protein; killer activating receptor associated protein;killer-activating receptor-

associated protein; etc.). For example, a suitable intracellularactivating domain can include a domain with at least 50%, 60%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to astretch of at least 10, 15, 20, or all amino acids in the followingsequences or to a contiguous stretch of from about 100 amino acids toabout 110 amino acids (aa), from about 110 aa to about 115 aa, fromabout 115 aa to about 120 aa, from about 120 aa to about 130 aa, fromabout 130 aa to about 140 aa, from about 140 aa to about 150 aa, or fromabout 150 aa to about 160 aa, of either

of the following amino acid sequences (4 isoforms):

-   -   MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLG        RLVPRGRGAAEAATRKQRITETESP[YQELQGQRSDVYSDL]NTQRPYYK (SEQ ID        NO:27),    -   MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLG        RLVPRGRGAAEATRKQRITETESP[YQELQGQRSDVYSDL]NTQ (SEQ ID NO:28),    -   MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAE        AATRKQRITETESP[YQELQGQRSDVYSDL]NTQRPYYK (SEQ ID NO:29), or    -   MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAE        ATRKQRITETESP[YQELQGQRSDVYSDL]NTQRPYYK (SEQ ID NO:30), where the        ITAM motifs are set out in brackets.

Likewise, a suitable intracellular activating domain polypeptide cancomprise an ITAM motif-containing portion of the full length DAP12 aminoacid sequence. Thus, a suitable intracellular activating domain caninclude a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least10, 15, 20, or all amino acids in the following sequence:ESP[YQELQGQRSDVYSDL]NTQ (SEQ ID NO:31), where the ITAM motifs are setout in brackets.

In some cases, the intracellular activating domain is derived fromFCERIG (also known as FCRG; Fc epsilon receptor I gamma chain; Fcreceptor gamma-chain; fc-epsilon RI-gamma; fcRgamma; fceRI gamma; highaffinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin Ereceptor, high affinity, gamma chain; etc.). For example, a suitableintracellular activating domain can include a domain with at least 50%,60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to a stretch of at least 10, 15, 20, or all amino acids in thefollowing sequences or to a contiguous stretch of from about 50 aminoacids to about 60 amino acids (aa), from about 60 aa to about 70 aa,from about 70 aa to about 80 aa, or from about 80 aa to about 88 aa,

of the following amino acid sequence:

-   -   MIPAVVLLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITSYEKSDGV[YT        GLSTRNQETYETL]KHEKPPQ (SEQ ID NO:32), where the ITAM motifs are        set out in brackets.

Likewise, a suitable intracellular activating domain polypeptide cancomprise an ITAM motif-containing portion of the full length FCER1Gamino acid sequence. Thus, a suitable intracellular activating domaincan include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of atleast 10, 15, 20, or all amino acids in the following sequence:DGV[YTGLSTRNQETYETL]KHE (SEQ ID NO:33), where the ITAM motifs are setout in brackets.

Intracellular activating domains suitable for use in an engineeredsignaling polypeptide of the present disclosure include a DAP10/CD28type signaling chain. An example of a DAP10 signaling chain is the aminoacid SEQ ID NO:34. In some embodiments, a suitable intracellularactivating domain includes a domain with at least 50%, 60%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to astretch of at least 10, 15, 20, or all amino acids in SEQ ID NO:34.

An example of a CD28 signaling chain is the amino acid sequence is SEQID NO:35. In some embodiments, a suitable intracellular domain includesa domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20,or all amino acids of SEQ ID NO:35.

Intracellular activating domains suitable for use in an engineeredsignaling polypeptide of the present disclosure include a ZAP70polypeptide, For example, a suitable intracellular activating domain caninclude a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least10, 15, 20, or all amino acids in the following sequences or to acontiguous stretch of from about 300 amino acids to about 400 aminoacids, from about 400 amino acids to about 500 amino acids, or fromabout 500 amino acids to 619 amino acids, of SEQ ID NO:36.

Modulatory Domains

Modulatory domains can change the effect of the intracellular activatingdomain in the engineered signaling polypeptide, including enhancing ordampening the downstream effects of the activating domain or changingthe nature of the response. Modulatory domains suitable for use in anengineered signaling polypeptide of the present disclosure includeco-stimulatory domains. A modulatory domain suitable for inclusion inthe engineered signaling polypeptide can have a length of from about 30amino acids to about 70 amino acids (aa), e.g., a modulatory domain canhave a length of from about 30 aa to about 35 aa, from about 35 aa toabout 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa,from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa. Inother cases, modulatory domain can have a length of from about 70 aa toabout 100 aa, from about 100 aa to about 200 aa, or greater than 200 aa.

Co-stimulatory domains typically enhance and/or change the nature of theresponse to an activation domain. Co-stimulatory domains suitable foruse in an engineered signaling polypeptide of the present disclosure aregenerally polypeptides derived from receptors. In some embodiments,co-stimulatory domains homodimerize. A subject co-stimulatory domain canbe an intracellular portion of a transmembrane protein (i.e., theco-stimulatory domain can be derived from a transmembrane protein).Non-limiting examples of suitable co-stimulatory polypeptides include,but are not limited to, 4-1BB (CD137), CD27, CD28, CD28 deleted for Lekbinding (ICΔ), ICOS, OX40, BTLA, CD27, CD30, GITR, and HVEM. Forexample, a co-stimulatory domain of an aspect of the invention can haveat least 80%, 90%, or 95% sequence identity to the co-stimulatory domainof 4-1BB (CD137), CD27, CD28, CD28 deleted for Lek binding (ICΔ), ICOS,OX40, BTLA, CD27, CD30, GITR, or HVEM. For example, a co-stimulatorydomain of an aspect of the invention can have at least 80%, 90%, or 95%sequence identity to the co-stimulatory domain of non-limiting examplesof suitable co-stimulatory polypeptides include, but are not limited to,4-1BB (CD137), CD27, CD28, CD28 deleted for Lek binding (ICΔ), ICOS,OX40, BTLA, CD27, CD30, GITR, and HVEM. For example, a co-stimulatorydomain of an aspect of the invention can have at least 80%, 90%, or 95%sequence identity to the co-stimulatory domain of 4-1BB (CD137), CD27,CD28, CD28 deleted for Lek binding (ICΔ), ICOS, OX40, BTLA, CD27, CD30,GITR, or HVEM.

A co-stimulatory domain suitable for inclusion in an engineeredsignaling polypeptide can have a length of from about 30 amino acids toabout 70 amino acids (aa), e.g., a co-stimulatory domain can have alength of from about 30 aa to about 35 aa, from about 35 aa to about 40aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa,from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, fromabout 60 aa to about 65 aa, or from about 65 aa to about 70 aa. In othercases, the co-stimulatory domain can have a length of from about 70 aato about 100 aa, from about 100 aa to about 200 aa, or greater than 200aa.

In some cases, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein CD137 (also known asTNFRSF9; CD137; 4-1BB; CDwl37; ILA; etc.). For example, a suitableco-stimulatory domain can include a domain with at least 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity toa stretch of at least 10, 15, 20, or all of the amino acids in SEQ IDNO:1. In some of these embodiments, the co-stimulatory domain has alength of from about 30 aa to about 35 aa, from about 35 aa to about 40aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa,from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, fromabout 60 aa to about 65 aa, or from about 65 aa to about 70 aa.

In some cases, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein CD28 (also known asTp44). For example, a suitable co-stimulatory domain can include adomain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20,or all of the amino acids in SEQ ID NO:2. In some of these embodiments,the co-stimulatory domain has a length of from about 30 aa to about 35aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa,from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, fromabout 55 aa to about 60 aa, from about 60 aa to about 65 aa, or fromabout 65 aa to about 70 aa.

In some cases, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein CD28 deleted for Lekbinding (ICΔ). For example, a suitable co-stimulatory domain can includea domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20,or all of the amino acids in SEQ ID NO:3. In some of these embodiments,the co-stimulatory domain has a length of from about 30 aa to about 35aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa,from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, fromabout 55 aa to about 60 aa, from about 60 aa to about 65 aa, or fromabout 65 aa to about 70 aa.

In some cases, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein ICOS (also known asAILIM, CD278, and CVIDl). For example, a suitable co-stimulatory domaincan include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of atleast 10, 15, 20, or all of the amino acids in SEQ ID NO:4. In some ofthese embodiments, the co-stimulatory domain has a length of from about30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aato about 45 aa, from about 45 aa to about 50 aa, from about 50 aa toabout 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about65 aa, or from about 65 aa to about 70 aa.

In some cases, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein OX40 (also known asTNFRSF4, RP5-902P8.3, ACT35, CD134, OX-40, TXGPIL). For example, asuitable co-stimulatory domain can include a domain with at least 50%,60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to a stretch of at least 10, 15, 20, or all of the amino acidsin SEQ ID NO:5. In some of these embodiments, the co-stimulatory domainhas a length of from about 30 aa to about 35 aa, from about 35 aa toabout 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa,from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.

In some cases, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein CD27 (also known as S152, T 14, TNFRSF7, and Tp55). For example, a suitable co-stimulatorydomain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch ofat least 10, 15, 20, or all of the amino acids in SEQ ID NO:6. In someof these embodiments, the co-stimulatory domain has a length of fromabout 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about40 aa to about 45 aa, from about 45 aa to about 50 aa, from about

In some cases, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein BTLA (also known asBTLAl and CD272). For example, a suitable co-stimulatory domain caninclude a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least10, 15, 20, or all of the amino acids in SEQ ID NO:7.

In some cases, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein CD30 (also known asTNFRSF8, D1S166E, and Ki-1). For example, a suitable co-stimulatorydomain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch offrom about 100 amino acids to about 110 amino acids (aa), from about 110aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aato about 130 aa, from about 130 aa to about 140 aa, from about 140 aa toabout 150 aa, from about 150 aa to about 160 aa, or from about 160 aa toabout 185 aa of SEQ ID NO:8.

In some cases, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein GITR (also known asTNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D). For example, a suitableco-stimulatory domain can include a domain with at least 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity toa stretch of at least 10, 15, 20, or all of the amino acids in SEQ IDNO:9. In some of these embodiments, the co-stimulatory domain has alength of from about 30 aa to about 35 aa, from about 35 aa to about 40aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa,from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, fromabout 60 aa to about 65 aa, or from about 65 aa to about 70 aa.

In some cases, the co-stimulatory domain derived from an intracellularportion of the transmembrane protein HVEM (also known as TNFRSF14,RP3-395M20.6, ATAR,

CD270, HVEA, HVEM, LIGHTR, and TR2). For example, a suitableco-stimulatory domain can include a domain with at least 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity toa stretch of at least 10, 15, 20, or all of the amino acids in SEQ IDNO:10. In some of these embodiments, the co-stimulatory domain of boththe first and the second polypeptide has a length of from about 30 aa toabout 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa,from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, orfrom about 65 aa to about 70 aa.

Linker

In some cases, the engineered signaling polypeptide includes a linkerbetween any two adjacent domains. For example, a linker can be betweenthe transmembrane domain and the first co-stimulatory domain. As anotherexample, the ASTR can be an antibody and a linker can be between theheavy chain and the light chain. As another example, a linker can bebetween the ASTR and the transmembrane domain and a co-stimulatorydomain. As another example, a linker can be between the co-stimulatorydomain and the intracellular activating domain of the secondpolypeptide. As another example, the linker can be between the ASTR andthe intracellular signaling domain.

The linker peptide may have any of a variety of amino acid sequences.Proteins can be joined by a spacer peptide, generally of a flexiblenature, although other chemical linkages are not excluded. A linker canbe a peptide of between about 1 and about 100 amino acids in length, orbetween about 1 and about 25 amino acids in length. These linkers can beproduced by using synthetic, linker-encoding oligonucleotides to couplethe proteins. Peptide linkers with a degree of flexibility can be used.The linking peptides may have virtually any amino acid sequence, bearingin mind that suitable linkers will have a sequence that results in agenerally flexible peptide. The use of small amino acids, such asglycine and alanine, are of use in creating a flexible peptide. Thecreation of such sequences is routine to those of skill in the art.

Suitable linkers can be readily selected and can be of any of a suitableof different lengths, such as from 1 amino acid (e.g., Gly) to 20 aminoacids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

Exemplary flexible linkers include glycine polymers (G)_(n),glycine-serine polymers (including, for example, (GS)_(n), GSGGS_(n),GGGS_(n), and GGGGS_(n) where n is an integer of at least one),glycine-alanine polymers, alanine-serine polymers, and other flexiblelinkers known in the art. Glycine and glycine-serine polymers are ofinterest since both of these amino acids are relatively unstructured,and therefore may serve as a neutral tether between components. Glycinepolymers are of particular interest since glycine accesses significantlymore phi-psi space than even alanine, and is much less restricted thanresidues with longer side chains (see Scheraga, Rev. Computational Chem.11173-142 (1992)). Exemplary flexible linkers include, but are notlimited GGGGSGGGGSGGGGS (SEQ ID NO:53), GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO:54), GGGGSGGGSGGGGS (SEQ ID NO:55), GGSG (SEQ ID NO:56),GGSGG (SEQ ID NO:57), GSGSG (SEQ ID NO:58), GSGGG (SEQ ID NO:59), GGGSG(SEQ ID NO:60), GSSSG (SEQ ID NO:61), and the like. The ordinarilyskilled artisan will recognize that design of a peptide conjugated toany elements described above can include linkers that are all orpartially flexible, such that the linker can include a flexible linkeras well as one or more portions that confer less flexible structure.

Combinations

In some embodiments, a polynucleotide provided by the replicationincompetent recombinant retroviral particles has one or moretranscriptional units that encode certain combinations of the one ormore engineered signaling polypeptides. In some methods and compositionsprovided herein, genetically modified T cells include the combinationsof the one or more engineered signaling polypeptides after transductionof T cells by the replication incompetent recombinant retroviralparticles. It will be understood that the reference of a firstpolypeptide, a second polypeptide, a third polypeptide, etc. is forconvenience and elements on a “first polypeptide” and those on a “secondpolypeptide” means that the elements are on different polypeptides thatare referenced as first or second for reference and convention only,typically in further elements or steps to that specific polypeptide.

In some embodiments, the first engineered signaling polypeptide includesan extracellular antigen binding domain, which is capable of binding anantigen, and an intracellular signaling domain. In other embodiments,the first engineered signaling polypeptide also includes a T cellsurvival motif and/or a transmembrane domain. In some embodiments, thefirst engineered signaling polypeptide does not include a co-stimulatorydomain, while in other embodiments, the first engineered signalingpolypeptide does include a co-stimulatory domain.

In some embodiments, a second engineered signaling polypeptide includesa lymphoproliferative gene product and optionally an extracellularantigen binding domain. In some embodiments, the second engineeredsignaling polypeptide also includes one or more of the following: a Tcell survival motif, an intracellular signaling domain, and one or moreco-stimulatory domains. In other embodiments, when two engineeredsignaling polypeptides are used, at least one is a CAR.

In one embodiment, the one or more engineered signaling polypeptides areexpressed under a T cell specific promoter or a general promoter underthe same transcript wherein in the transcript, nucleic acids encodingthe engineered signaling polypeptides are separated by nucleic acidsthat encode one or more internal ribosomal entry sites (IREs) or one ormore protease cleavage peptides.

In certain embodiments, the polynucleotide encodes two engineeredsignaling polypeptides wherein the first engineered signalingpolypeptide includes a first extracellular antigen binding domain, whichis capable of binding to a first antigen, and a first intracellularsignaling domain but not a co-stimulatory domain, and the secondpolypeptide includes a second extracellular antigen binding domain,which is capable of binding VEGF, and a second intracellular signalingdomain, such as for example, the signaling domain of a co-stimulatorymolecule. In a certain embodiment, the first antigen is PSCA, PSMA, orBCMA. In a certain embodiment, the first extracellular antigen bindingdomain comprises an antibody or fragment thereof (e.g., scFv), e.g., anantibody or fragment thereof specific to PSCA, PSMA, or BCMA. In acertain embodiment, the second extracellular antigen binding domain thatbinds VEGF is a receptor for VEGF, i.e., VEGFR. In certain embodiments,the VEGFR is VEGFR1, VEGFR2, or VEGFR3. In a certain embodiment, theVEGFR is VEGFR2.

In certain embodiments, the polynucleotide encodes two engineeredsignaling polypeptides wherein the first engineered signalingpolypeptide includes an extracellular tumor antigen binding domain and aCD3ζ signaling domain, and the second engineered signaling polypeptideincludes an antigen-binding domain, wherein the antigen is an angiogenicor vasculogenic factor, and one or more co-stimulatory moleculesignaling domains. The angiogenic factor can be, e.g., VEGF. The one ormore co-stimulatory molecule signaling motifs can comprise, e.g.,co-stimulatory signaling domains from each of CD27, CD28, OX40, ICOS,and 4-1BB.

In certain embodiments, the polynucleotide encodes two engineeredsignaling polypeptides wherein the first engineered signalingpolypeptide includes an extracellular tumor antigen-binding domain and aCD3ζ signaling domain, the second polypeptide comprises anantigen-binding domain, which is capable of binding to VEGF, andco-stimulatory signaling domains from each of CD27, CD28, OX40, ICOS,and 4-1BB. In a further embodiment, the first signaling polypeptide orsecond signaling polypeptide also has a T cell survival motif. In someembodiments, the T cell survival motif is, or is derived from, anintracellular signaling domain of IL-7 receptor (IL-7R), anintracellular signaling domain of IL-12 receptor, an intracellularsignaling domain of IL-15 receptor, an intracellular signaling domain ofIL-21 receptor, or an intracellular signaling domain of transforminggrowth factor β (TGFβ) receptor or the TGFβ decoy receptor(TGF-β-dominant-negative receptor II (DNRII)).

In certain embodiments, the polynucleotide encodes two engineeredsignaling polypeptides wherein the first engineered signalingpolypeptide includes an extracellular tumor antigen-binding domain and aCD3ζ signaling domain, and the second engineered signaling polypeptideincludes an antigen-binding domain, which is capable of binding to VEGF,an IL-7 receptor intracellular T cell survival motif, and co-stimulatorysignaling domains from each of CD27, CD28, OX40, ICOS, and 4-1BB.

In some embodiments, more than two signaling polypeptides are encoded bythe polynucleotide. In certain embodiments, only one of the engineeredsignaling polypeptides includes an antigen binding domain that binds toa tumor-associated antigen or a tumor-specific antigen; each of theremainder of the engineered signaling polypeptides comprises an antigenbinding domain that binds to an antigen that is not a tumor-associatedantigen or a tumor-specific antigen. In other embodiments, two or moreof the engineered signaling polypeptides include antigen binding domainsthat bind to one or more tumor-associated antigens or tumor-specificantigens, wherein at least one of the engineered signaling polypeptidescomprises an antigen binding domain that does not bind to atumor-associated antigen or a tumor-specific antigen.

In some embodiments, the tumor-associated antigen or tumor-specificantigen is Her2, prostate stem cell antigen (PSCA), PSMA(prostate-specific membrane antigen), B cell maturation antigen (BCMA),alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancerantigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membraneprotein (EMA), epithelial tumor antigen (ETA), tyrosinase,melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117,chromogranin, cytokeratin, desmin, glial fibrillary acidic protein(GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen,protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1),myo-D1, muscle-specific actin (MSA), neurofilament, neuron-specificenolase (NSE), placental alkaline phosphatase, synaptophysin,thyroglobulin, thyroid transcription factor-1, the dimeric form of thepyruvate kinase isoenzyme type M2 (tumor M2-PK), CD19, CD22, CD27, CD30,CD70, GD2 (ganglioside G2), EphA2, CSPG4, CD138, FAP (FibroblastActivation Protein), CD171, kappa, lambda, 5T4, αvβ6 integrin, integrinαvβ3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viraloncogene), Ral-B, B7-H3, B7-H6, CAIX, CD20, CD33, CD44, CD44v6,CD44v7/8, CD123, EGFR, EGP2, EGP40, EpCAM, fetal AchR, FRα, GD3,HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-IIRα, IL-13Rα2, Lewis-Y, Muc16, NCAM,NKG2D Ligands, NY-ESO-1, PRAME, ROR1, Survivin, TAG72, TEMs, VEGFR2,EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Sp17),mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cellreceptor gamma alternate reading frame protein), Trp-p8, STEAP1(six-transmembrane epithelial antigen of the prostate 1), an abnormalras protein, or an abnormal p53 protein.

In some embodiments, the first engineered signaling polypeptide includesa first extracellular antigen binding domain that binds a first antigen,and a first intracellular signaling domain; and a second engineeredsignaling polypeptide includes a second extracellular antigen bindingdomain that binds a second antigen, or a receptor that binds the secondantigen; and a second intracellular signaling domain, wherein the secondengineered signaling polypeptide does not comprise a co-stimulatorydomain. In a certain embodiment, the first antigen-binding domain andthe second antigen-binding domain are independently an antigen-bindingportion of a receptor or an antigen-binding portion of an antibody. In acertain embodiment, either or both of the first antigen binding domainor the second antigen binding domain are scFv antibody fragments. Incertain embodiments, the first engineered signaling polypeptide and/orthe second engineered signaling polypeptide additionally comprises atransmembrane domain. In a certain embodiment, the first engineeredsignaling polypeptide or the second engineered signaling polypeptidecomprises a T cell survival motif, e.g., any of the T cell survivalmotifs described herein.

In another embodiment, the first engineered signaling polypeptideincludes a first extracellular antigen binding domain that binds HER2and the second engineered signaling polypeptide includes a secondextracellular antigen binding domain that binds MUC-1.

In another embodiment, the second extracellular antigen binding domainof the second engineered signaling polypeptide binds an interleukin.

In another embodiment, the second extracellular antigen binding domainof the second engineered signaling polypeptide binds a damage associatedmolecular pattern molecule (DAMP; also known as an alarmin). In otherembodiments, a DAMP is a heat shock protein, chromatin-associatedprotein high mobility group box 1 (HMGB1), S100A8 (also known as MRP8,or calgranulin A), S100A9 (also known as MRP14, or calgranulin B), serumamyloid A (SAA), deoxyribonucleic acid, adenosine triphosphate, uricacid, or heparin sulfate.

In certain embodiments, said second antigen is an antigen on an antibodythat binds to an antigen presented by a tumor cell.

In some embodiments, signal transduction activation through the secondengineered signaling polypeptide is non-antigenic, but is associatedwith hypoxia. In certain embodiments, hypoxia is induced by activationof hypoxia-inducible factor-1α (HIF-1α), HIF-1β, HIF-2α, HIF-2β, HIF-3α,or HIF-3β.

In some embodiments, expression of the one or more engineered signalingpolypeptides is regulated by a control element, which is disclosed inmore detail herein.

Additional Sequences

The engineered signaling polypeptides, such as CARs, can further includeone or more additional polypeptide domains, where such domains include,but are not limited to, a signal sequence; an epitope tag; an affinitydomain; and a polypeptide whose presence or activity can be detected(detectable marker), for example by an antibody assay or because it is apolypeptide that produces a detectable signal. Non-limiting examples ofadditional domains for any of the aspects or embodiments providedherein, include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of thefollowing sequences as described below: a signal sequence, an epitopetag, an affinity domain, or a polypeptide that produces a detectablesignal.

Signal sequences that are suitable for use in a subject CAR, e.g., inthe first polypeptide of a subject CAR, include any eukaryotic signalsequence, including a naturally-occurring signal sequence, a synthetic(e.g., man-made) signal sequence, etc. In some embodiments, for example,the signal sequence can be the CD8 signal sequence MALPVTALLLPLALLLHAARP(SEQ ID NO:74).

Suitable epitope tags include, but are not limited to, hemagglutinin(HA; e.g., YPYDVPDYA; SEQ ID NO:37); FLAG (e.g., DYKDDDDK; SEQ IDNO:38); c-myc (e.g., EQKLISEEDL; SEQ ID NO:39), and the like.

Affinity domains include peptide sequences that can interact with abinding partner, e.g., such as one immobilized on a solid support,useful for identification or purification. DNA sequences encodingmultiple consecutive single amino acids, such as histidine, when fusedto the expressed protein, may be used for one-step purification of therecombinant protein by high affinity binding to a resin column, such asnickel sepharose. Exemplary affinity domains include His5 (HHHHH; SEQ IDNO:40), HisX6 (HHHHHH; SEQ ID NO:41), c-myc (EQKLISEEDL; SEQ ID NO:39),Flag (DYKDDDDK; SEQ ID NO:38), Strep Tag (WSHPQFEK; SEQ ID NO:42),hemagglutinin, e.g., HA Tag (YPYDVPDYA; SEQ ID NO:37), GST, thioredoxin,cellulose binding domain, RYIRS (SEQ ID NO:43), Phe-His-His-Thr (SEQ IDNO:44), chitin binding domain, S-peptide, T7 peptide, SH2 domain, C-endRNA tag, WEAAAREACCRECCARA (SEQ ID NO:45), metal binding domains, e.g.,zinc binding domains or calcium binding domains such as those fromcalcium-binding proteins, e.g., calmodulin, troponin C, calcineurin B,myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin,hippocalcin, frequenin, caltractin, calpain large-subunit, S100proteins,parvalbumin, calbindin D9K, calbindin D28K, and calretinin, inteins,biotin, streptavidin, MyoD, Id, leucine zipper sequences, and maltosebinding protein.

Suitable detectable signal-producing proteins include, e.g., fluorescentproteins; enzymes that catalyze a reaction that generates a detectablesignal as a product; and the like.

Suitable fluorescent proteins include, but are not limited to, greenfluorescent protein (GFP) or variants thereof, blue fluorescent variantof GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescentvariant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhancedYFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine,GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP),destabilized EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet,mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2,t-dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP,Kaede protein and kindling protein, Phycobiliproteins andPhycobiliprotein conjugates including B-Phycoerythrin, R-Phycoerythrinand Allophycocyanin. Other examples of fluorescent proteins includemHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry,mCherry, mGrapel

mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods2:905-909), and the like. Any of a variety of fluorescent and coloredproteins from Anthozoan species, as described in, e.g., Matz et al.(1999) Nature Biotechnol. 17:969-973, is suitable for use.

Suitable enzymes include, but are not limited to, horse radishperoxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL),glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase,β-glucuronidase, invertase, Xanthine Oxidase, firefly luciferase,glucose oxidase (GO), and the like.

Recognition and/or Elimination Domain

Any of the replication incompetent recombinant retroviral particlesprovided herein can include nucleic acids that encode a recognition orelimination domain as part of, or separate from, nucleic acids encodingany of the engineered signaling polypeptides provided herein. Thus, anyof the engineered signaling polypeptides provided herein, can include arecognition or elimination domain. For example, any of the CARsdisclosed herein can include a recognition or elimination domain.Moreover, a recognition or elimination domain can be expressed togetherwith, or even fused with any of the lymphoproliferative elementsdisclosed herein. The recognition or elimination domains are expressedon the T cell and/or NK cell but are not expressed on the replicationincompetent recombinant retroviral particles.

In some embodiments, the recognition or elimination domain can bederived from herpes simplex virus-derived enzyme thymidine kinase(HSV-tk) or inducible caspase-9. In some embodiments, the recognition orelimination domain can include a modified endogenous cell-surfacemolecule, for example as disclosed in U.S. Pat. No. 8,802,374. Themodified endogenous cell-surface molecule can be any cell-surfacerelated receptor, ligand, glycoprotein, cell adhesion molecule, antigen,integrin, or cluster of differentiation (CD) that is modified. In someembodiments, the modified endogenous cell-surface molecule is atruncated tyrosine kinase receptor. In one aspect, the truncatedtyrosine kinase receptor is a member of the epidermal growth factorreceptor (EGFR) family (e.g., ErbB1, ErbB2, ErbB3, and ErbB4). In someembodiments, the recognition domain can be a polypeptide that isrecognized by an antibody that recognizes the extracellular domain of anEGFR member. In some embodiments, the recognition domain can be at least20 contiguous amino acids of an EGFR family member, or for example,between 20 and 50 contiguous amino acids of an EGFR family member. Forexample, SEQ ID NO:78, is an exemplary polypeptide that is recognizedby, and under the appropriate conditions bound by an antibody thatrecognizes the extracellular domain of an EGFR member. Suchextracellular EGFR epitopes are sometimes referred to herein as eTags.In illustrative embodiments, such epitopes are recognized bycommercially available anti-EGFR monoclonal antibodies.

Epidermal growth factor receptor, also known as EGFR, ErbB1 and HER1, isa cell-surface receptor for members of the epidermal growth factorfamily of extracellular ligands. Alterations in EGFR activity have beenimplicated in certain cancers. In some embodiments, a gene encoding anEGFR polypeptide including human epidermal growth factor receptor (EGFR)is constructed by removal of nucleic acid sequences that encodepolypeptides including the membrane distal EGF-binding domain and thecytoplasmic signaling tail, but retains the extracellular membraneproximal epitope recognized by an anti-EGFR antibody. Preferably, theantibody is a known, commercially available anti-EGFR monoclonalantibody, such as cetuximab, matuzumab, necitumumab or panitumumab.

Others have shown that application of biotinylated-cetuximab toimmunomagnetic selection in combination with anti-biotin microbeadssuccessfully enriches T cells that have been lentivirally transducedwith EGFRt-containing constructs from as low as 2% of the population togreater than 90% purity without observable toxicity to the cellpreparation. Furthermore, others have shown that constitutive expressionof this inert EGFR molecule does not affect T cell phenotype or effectorfunction as directed by the coordinately expressed chimeric antigenreceptor (CAR), CD19R. In addition, others have shown that through flowcytometric analysis, EGFR was successfully utilized as an in vivotracking marker for T cell engraftment in mice. Furthermore, EGFR wasdemonstrated to have suicide gene potential through Erbitux® mediatedantibody dependent cellular cytotoxicity (ADCC) pathways. The inventorsof the present disclosure have successfully expressed eTag in PBMCsusing lentiviral vectors, and have found that expression of eTag invitro by PBMCs exposed to Cetuximab, provided an effective eliminationmechanism for PBMCs. Thus, EGFR may be used as a non-immunogenicselection tool, tracking marker, and suicide gene for transduced T cellsthat have immunotherapeutic potential. The EGFR nucleic acid may also bedetected by means well known in the art.

In some embodiments provided herein, EGFR is expressed as part of asingle polypeptide that also includes the CAR or as part of a singlepolypeptide that includes the lymphoproliferative element. In someembodiments, the amino acid sequence encoding the EGFR recognitiondomain can be separated from the amino acid sequence encoding thechimeric antigen receptor by a cleavage signal and/or a ribosomal skipsequence. The ribosomal skip and/or cleavage signal can be any ribosomalskip and/or cleavage signal known in the art. Not to be limited bytheory, the ribosomal skip sequence can be, for example T2A (alsoreferred to as 2A-1 herein) with amino acid sequenceGSGEGRGSLLTCGDVEENPGP (SEQ ID NO:77). Not to be limited by theory, otherexamples of cleavage signals and ribosomal skip sequences include FMDV2A (F2A); equine rhinitis A virus 2A (abbreviated as E2A); porcineteschovirus-1 2A (P2A); and Thoseaasigna virus 2A (T2A). In someembodiments, the polynucleotide sequence encoding the recognition domaincan be on the same transcript as the CAR or lymphoproliferative elementbut separated from the polynucleotide sequence encoding the CAR orlymphoproliferative element by an internal ribosome entry site.

In other embodiments as exemplified empirically herein, a recognitiondomain can be expressed as part of a fusion polypeptide, fused to alymphoproliferative element. Such constructs provide the advantage,especially in combination with other “space saving” elements providedherein, of taking up less genomic space on an RNA genome compared toseparate polypeptides. In one illustrative embodiment, an eTag isexpressed as a fusion polypeptide, fused to an IL7Rα mutant, asexperimentally demonstrated herein.

Chimeric Antigen Receptor

In some aspects of the present invention, an engineered signalingpolypeptide is a chimeric antigen receptor (CAR) or a polynucleotideencoding a CAR, which, for simplicity, is referred to herein as “CAR.” ACAR of the present disclosure includes: a) at least one antigen-specifictargeting region (ASTR); b) a transmembrane domain; and c) anintracellular activating domain. In illustrative embodiments, theantigen-specific targeting region of the CAR is a scFv portion of anantibody to the target antigen. In illustrative embodiments, theintracellular activating domain is from CD3z.

A CAR of the present disclosure can be present in the plasma membrane ofa eukaryotic cell, e.g., a mammalian cell, where suitable mammaliancells include, but are not limited to, a cytotoxic cell, a T lymphocyte,a stem cell, a progeny of a stem cell, a progenitor cell, a progeny of aprogenitor cell, and an NK cell, an NK-T cell, and a macrophage. Whenpresent in the plasma membrane of a eukaryotic cell, a CAR of thepresent disclosure is active in the presence of one or more targetantigens that, in certain conditions, binds the ASTR. The target antigenis the second member of the specific binding pair. The target antigen ofthe specific binding pair can be a soluble (e.g., not bound to a cell)factor; a factor present on the surface of a cell such as a target cell;a factor presented on a solid surface; a factor present in a lipidbilayer; and the like. Where the ASTR is an antibody, and the secondmember of the specific binding pair is an antigen, the antigen can be asoluble (e.g., not bound to a cell) antigen; an antigen present on thesurface of a cell such as a target cell; an antigen presented on a solidsurface; an antigen present in a lipid bilayer; and the like.

In some instances, a CAR of the present disclosure, when present in theplasma membrane of a eukaryotic cell, and when activated by one or moretarget antigens, increases expression of at least one nucleic acid inthe cell. For example, in some cases, a CAR of the present disclosure,when present in the plasma membrane of a eukaryotic cell, and whenactivated by the one or more target antigens, increases expression of atleast one nucleic acid in the cell by at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 40%, at least about 50%, at least about 75%, at least about2-fold, at least about 2.5-fold, at least about 5-fold, at least about10-fold, or more than 10-fold, compared with the level of transcriptionof the nucleic acid in the absence of the one or more target antigens.

As an example, the CAR of the present disclosure can include animmunoreceptor tyrosine-based activation motif (ITAM)-containingintracellular signaling polypeptide.

A CAR of the present disclosure, when present in the plasma membrane ofa eukaryotic cell, and when activated by one or more target antigens,can, in some instances, result in increased production of one or morecytokines by the cell. For example, a CAR of the present disclosure,when present in the plasma membrane of a eukaryotic cell, and whenactivated by the one or more target antigens, can increase production ofa cytokine by the cell by at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about40%, at least about 50%, at least about 75%, at least about 2-fold, atleast about 2.5-fold, at least about 5-fold, at least about 10-fold, ormore than 10-fold, compared with the amount of cytokine produced by thecell in the absence of the one or more target antigens. Cytokines whoseproduction can be increased include, but are not limited to interferongamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), IL-2, IL-15, IL-12,IL-4, IL-5, IL-10; a chemokine; a growth factor; and the like.

In some cases, a CAR of the present disclosure, when present in theplasma membrane of a eukaryotic cell, and when activated by one or moretarget antigens, can result in both an increase in transcription of anucleic acid in the cell and an increase in production of a cytokine bythe cell.

In some instances, a CAR of the present disclosure, when present in theplasma membrane of a eukaryotic cell, and when activated by one or moretarget antigens, results in cytotoxic activity by the cell toward atarget cell that expresses on its cell surface an antigen to which theantigen-binding domain of the first polypeptide of the CAR binds. Forexample, where the eukaryotic cell is a cytotoxic cell (e.g., an NK cellor a cytotoxic T lymphocyte), a CAR of the present disclosure, whenpresent in the plasma membrane of the cell, and when activated by theone or more target antigens, increases cytotoxic activity of the celltoward a target cell that expresses on its cell surface the one or moretarget antigens. For example, where the eukaryotic cell is an NK cell ora T lymphocyte, a CAR of the present disclosure, when present in theplasma membrane of the cell, and when activated by the one or moretarget antigens, increases cytotoxic activity of the cell by at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 40%, at least about 50%, at leastabout 75%, at least about 2-fold, at least about 2.5-fold, at leastabout 5-fold, at least about 10-fold, or more than 10-fold, compared tothe cytotoxic activity of the cell in the absence of the one or moretarget antigens.

In some cases, a CAR of the present disclosure, when present in theplasma membrane of a eukaryotic cell, and when activated by one or moretarget antigens, can result in other CAR activation related events suchas proliferation and expansion (either due to increased cellulardivision or anti-apoptotic responses).

In some cases, a CAR of the present disclosure, when present in theplasma membrane of a eukaryotic cell, and when activated by one or moretarget antigens, can result in other CAR activation related events suchas intracellular signaling modulation, cellular differentiation, or celldeath.

In some embodiments, CARs of the present disclosure are microenvironmentrestricted. This property is typically the result of themicroenvironment restricted nature of the ASTR domain of the CAR. Thus,CARs of the present disclosure can have a lower binding affinity or, inillustrative embodiments, can have a higher binding affinity to one ormore target antigens under a condition(s) in a microenvironment thanunder a condition in a normal physiological environment.

Lymphoproliferative Elements

Peripheral T lymphocyte numbers are maintained at remarkably stablelevels throughout adulthood, despite the continuing addition of cells,due to emigration from the thymus and proliferation in response toantigen encounter, and loss of cells owing to the removal ofantigen-specific effectors after antigen clearance (Marrak, P. et al.2000. Nat Immunol 1:107-111; Freitas, A. A. et al. 2000. Annu RevImmunol 18:83-111). The size of the peripheral T cell compartment isregulated by multiple factors that influence both proliferation andsurvival. However, in a lymphopenic environment, T lymphocytes divideindependently of cognate antigen, due to “acute homeostaticproliferation” mechanisms that maintain the size of the peripheral Tcell compartment. Conditions for lymphopenia have been established insubjects or patients during adoptive cell therapy by proliferating Tcells in vitro and introducing them into lymphodepleted subjects,resulting in enhanced engraftment and antitumor function of transferredT cells. However, lymphodepletion of a subject is not desirable becauseit can cause serious side effects, including immune dysfunction anddeath.

Studies have shown that lymphodepletion removes endogenous lymphocytesfunctioning as cellular sinks for homeostatic cytokines, thereby freeingcytokines to induce survival and proliferation of adoptively transferredcells. Some cytokines, such as for example, IL-7 and IL-15, are known tomediate antigen-independent proliferation of T cells and are thuscapable of eliciting homeostatic proliferation in non-lymphopenicenvironments. However, these cytokines and their receptors haveintrinsic control mechanisms that prevent lymphoproliferative disordersat homeostasis.

Many of the aspects provided herein include a lymphoproliferativeelement, or a nucleic acid encoding the same, typically as part of anengineered signaling polypeptide. Accordingly, in some aspects of thepresent invention, an engineered signaling polypeptide is alymphoproliferative element such as a chimeric lymphoproliferativeelement (CLE). Typically, the CLE contains an extracellular domain, atransmembrane domain, and at least one intracellular signaling domainthat drives proliferation. A CLE does not comprise both an ASTR and anactivation domain. In illustrative embodiments herein, one or morelymphoproliferative elements is introduced into a resting T cell and/orresting NK cell, typically by transducing the resting T cell and/orresting NK cell with replication incompetent recombinant retroviralparticles whose genome encodes the lymphoproliferative element as partof an engineered signaling polypeptide. The lymphoproliferative elementcan include a single intracellular domain or can include more than oneintracellular domain. In certain illustrative embodiments, alymphoproliferative element includes two intracellular domains. Tables7-23 provided herein and discussed in Examples 17 and 18 provide CLEswith one intracellular domain and CLEs with two intracellular domainsthat were identified using experimental methods provided therein thattested candidate chimeric polypeptides that included differentcombinations of extracellular domains and intracellular domains that areidentified in Table 6.

In some illustrative embodiments, a lymphoproliferative element can beor can comprise a cytokine or in further illustrative embodiments, acytokine receptor, or a fragment that includes a signaling domainthereof, that activates a STAT3 pathway, a STAT4 pathway, or in evenfurther illustrative embodiments, a Jak/STAT5 pathway. As such, alymphoproliferative element, can be, in a non-limiting example, acytokine receptor, or active fragment that includes a signaling domainthereof, such as an interleukin receptor, or an active fragment thatincludes a signaling domain thereof, that activates STAT5. Thus, alymphoproliferative element is a polypeptide that promotesproliferation, and optionally survival (anti-apoptotic), and optionallyprovides a co-stimulatory signal that enhances a differentiation state,proliferative potential or resistance to cell death of a lymphocyte. Incertain illustrative embodiments, a lymphoproliferative element is apolypeptide that induces proliferation of a T cell and/or NK cell.Illustrative lymphoproliferative elements induce proliferation byactivating STAT5. Thus, fragments of such lymphoproliferative elementsretain the ability to induce proliferation of T cells and/or NK cells,in illustrative embodiments, by activating STAT5.

In illustrative embodiments, lymphoproliferative elements when presentin genetically modified PBMCs, lymphocytes, or genetically modified Tcells and/or NK cells are capable of promoting lymphocyteproliferation/expansion and optionally survival ex vivo or in vitro inculture in the absence of exposure of the cells to cytokines such asIL-15, IL-7, and in illustrative embodiments IL-2 and the target for anASTR of a CAR expressed by the cells during culturing for 6, 7, 14, 21,or 35 days.

In some of the methods and compositions presented herein, alymphoproliferative element is used to promote proliferation orexpansion of genetically modified T cells in vivo without having tolymphodeplete subjects. As such, non-limiting illustrative embodimentsof methods provided herein that include inserting a lymphoproliferativeelement into a resting T cell and/or NK cell of a subject, typically bytransducing such T cell and/or NK cell can be performed withoutlymphodepleting the subject before, during and/or after performing themethod, or without lymphodepleting the subject before, during and/orafter collecting blood from a subject before performing such method, orwithout lymphodepleting the subject before, during, and/or aftergenetically modifying T cells or NK cells ex vivo from the subject,and/or before, during, or after reintroducing the genetically modified Tcells and/or NK cells into the subject. Factors that promoteproliferation of T cells in vivo include cytokines and their receptors,in which a receptor typically includes a ligand binding domain and asignaling domain. In some embodiments, the lymphoproliferative elementused in the methods and compositions disclosed herein is a cytokineand/or a cytokine receptor. The cytokine can be an interleukin, and thecytokine receptor can be an interleukin receptor. Thelymphoproliferative element can be a functional fragment of a cytokineand/or a functional fragment of a cytokine receptor, such as a signalingdomain thereof, wherein the fragment is capable of promotingproliferation of T cells, for example by activating STAT5.

In some embodiments, the cytokine lymphoproliferative element in themethods and compositions herein include one or more of the following:Interleukin-7 (IL-7) or its receptor (IL-7R), or a signaling domainthereof; Interleukin-12 (IL-12) or its receptor (IL-12R), or a signalingdomain thereof; Interleukin-23 (IL-23) or its receptor composed ofIL-12R β1 and IL-23R, or a signaling domain thereof; Interleukin-27(IL-27) or its receptor (IL-27R), or a signaling domain thereof;Interleukin-15 (IL-15) or its receptor (IL-15R), or a signaling domainthereof; Interleukin-21 (IL-21) or its receptor (IL-21R), or a signalingdomain thereof; or transforming growth factor β (TGFβ) or its receptor(TGFβR) or a signaling domain thereof; or the TGFβ decoy receptor(TGF-β-dominant-negative receptor II (DNRII)). In some embodiments, thelymphoproliferative element is the IL-12R or the TGFβ decoy receptor(TGF-β-dominant-negative receptor II (DNRII)).

IL-7 binds to the IL-7 receptor, a heterodimer consisting of IL-7R alphaand common gamma chain receptor. Binding results in a cascade of signalsimportant for T cell development within the thymus and survival withinthe periphery. Binding of IL-7 to the IL-7 receptor is known to activatethe Jak/STAT5 pathway.

IL-12 is involved in the differentiation of naïve T cells into Th1 cells(Hsieh C S et al. 1993. Science. 260(5107):547-9) and is known as a Tcell-stimulating factor. IL-12 binds to the IL-12 receptor, which is aheterodimeric receptor formed by IL-12R-01 and IL-12R-02. IL12 can actby activating STAT4, but has been shown to activate STAT5 in T cells aswell (Ahn, H., et al. 1998. J. Immun. 161:5893-5900). The IL-12 familyis composed of the cytokines IL-12, IL-23, and IL-27. The receptor forIL-23 is composed of IL-12R β1 and IL-23R. IL-27 is a heterodimericcytokine that is composed of two distinct genes, Epstein-Barrvirus-induced gene 3 (EBI3) and IL-27p28. IL-27 interacts with IL-27receptor.

IL-15 is a T and NK cell stimulatory factor that is similar in structureand function to IL-2. Both cytokines induce proliferation of T cells;and their shared functions are thought to result from both receptorsusing the IL-2/IL-15RD and common γ chains. Signaling pathway of IL-15begins with binding to IL-15Rα receptor, with subsequent presentation tosurrounding cells bearing IL-15Rβγc complex on their cell surface. Uponbinding IL-150 subunit activates Janus kinase 1 (Jak1) and γc subunitJanus kinase 3 (Jak3), which leads to phosphorylation and activation ofSTAT3 and STAT5.

IL-21 is expressed in activated human CD4+ T cells and in NKT cells, andIL-21 expression is up-regulated in Th2 and Th17 subsets of T helpercells. The IL-21 receptor (IL-21R) is expressed on the surface of T, Band NK cells and is similar in structure to the receptors for other typeI cytokines like IL-2R or IL-15. IL-21R requires dimerization with thecommon gamma chain (γc) in order to bind IL-21. When bound to IL-21, theIL-21 receptor acts through the Jak/STAT pathway, activating STAT1,STAT3, and STAT5.

TGFβ decoy receptors (TGF-β-dominant-negative receptor II (DNRII)) blockTGFβ signaling by competing with the natural receptors for TGFβ binding.TGFβ-DNRII is a kinase-dead truncated form of RII that contains theextracellular TGFβ binding domain and the transmembrane domain of RI.TGFβ-DNRII binds the ligand but does not phosphorylate and activate RI,which thereby diminishes or eliminates Smad phosphorylation.

Gain-of-function mutations in IL-7Rα have been identified in subjectswith B and T cell acute lymphoblastic leukemias (B-ALL and T-ALL)(Zenatti P P, et al. 2011. Nat Genet 43:932-939; Snochat, C. et al.2011. J Exp Med 208:901-908; McElroy, C. A. et al. 2012. PNAS109(7):2503-2508). The mutations included insertions and deletions inthe N-terminal region of the IL-7Rα TMD, with nearly all of thesequences containing an extra Cys residue, and an 5165-to-C165 mutation.The cysteine resulted in constitutive activation of the receptor. Someof the mutations in the T-all group activated JAK1. Thesegain-of-function IL-7R mutants can be used in any of the aspectsprovided herein as one of the lymphoproliferative element(s).

Accordingly, in some embodiments, the lymphoproliferative element is amutated IL-7 receptor. In other embodiments, the mutated IL-7 receptoris constitutively active, activating the JAK-STAT5 pathway in theabsence of the cytokine ligand. In still other embodiments, the mutatedIL-7 receptor comprises a 1 to 10 amino acid insertion at a positionbetween 237 and 254 that includes a cysteine residue that includes theability to constitutively activate the STAT5 pathway. In someembodiments, the mutated IL-7 receptor is IL-7Rα-insPPCL (represented bySEQ ID NO:82). Furthermore, in some chimeric lymphoproliferative element(CLE) embodiments provided herein, one or more, but not all, of thedomains is from IL-7Rα-insPPCL.

In some embodiments, the lymphoproliferative element is a chimericcytokine receptor such as but not limited to a cytokine tethered to itsreceptor that typically constitutively activates the same STAT pathwayas a corresponding activated wild-type cytokine receptor such as STAT3,STAT4, and in illustrative embodiments, STAT5. In some embodiments, thechimeric cytokine receptor is an interleukin, or a fragment thereof,tethered to or covalently attached to its cognate receptor, or afragment thereof, via a linker. In some embodiments, the chimericcytokine receptor is IL-7 tethered to IL-7Rα. In other embodiments, thechimeric cytokine receptor is IL-7 tethered to a domain of IL-7Rα, suchas for example, the extracellular domain of IL-7Rα and/or thetransmembrane domain of IL-7Rα. In some embodiments, thelymphoproliferative element is a cytokine receptor that is not tetheredto a cytokine, and in fact in illustrative embodiments, provided hereina lymphoproliferative element is a constitutively active cytokinereceptor that is not tethered to a cytokine. These chimeric IL-7receptors typically constitutively activate STAT5 when expressed.

In illustrative embodiments of any of the methods and compositionsprovided herein that include a lymphoproliferative element, wherein thelymphoproliferative element is a cytokine or cytokine receptorpolypeptide, or a fragment thereof comprising a signaling domain, thelymphoproliferative element can comprise an interleukin polypeptidecovalently attached to a portion of its cognate interleukin receptorpolypeptide via a linker. Typically, this portion of the cognateinterleukin receptor includes a functional portion of the extracellulardomain capable of binding the interleukin cytokine and the transmembranedomain. In some embodiments, the intracellular domain is anintracellular portion of the cognate interleukin receptor. In someembodiments, the intracellular domain is an intracellular portion of adifferent cytokine receptor that is capable of promoting lymphocyteproliferation. In some embodiments the lymphoproliferative element is aninterleukin polypeptide covalently attached to its full length cognateinterleukin receptor polypeptide via a linker.

In some embodiments, the lymphoproliferative element can include acytokine receptor or a fragment that includes a signaling domainthereof. In some embodiments, the cytokine receptor can be CD40, CRLF2,CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2,IFNLR1, IL1R1, ILIRAP, IL1RL1, IL1RL2, IL2RA, IL2RG, IL2RB, IL3RA, IL4R,IL5RA, IL6R, IL6ST, IL7RA, IL9R, IL10RA, IL10RB, IL12RB1, IL13RA2,IL15RA, IL17RA, IL17RB, IL17RC, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB,IL21R, IL22RA1, IL23R, IL27R, IL31RA, LEPR, LIFR, MPL, OSMR, PRLR,TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, or TNFRSF18. Exemplary embodimentsof chimeric cytokine receptors that include functional intracellulardomains of the above-listed cytokine receptors were tested in Examples17 and 18 to confirm that chimeric cytokine receptors that includedthese functional intracellular domains could function aslymphoproliferative elements.

In certain illustrative embodiments, the lymphoproliferative elementcomprises a cytokine receptor, or a fragment thereof that includes asignaling domain, that activates a Jak/STAT5 pathway. For example, suchlymphoproliferative element can include an intracellular domain ofIL21R, IL27R, IL31RA, LIFR, and OSMR. As shown in the experiments ofExamples 17 and 18 and Tables 7 to 17, chimeric lymphoproliferativeelements that include intracellular domains of these genes were foundamong the candidate chimeric polypeptides that induced the highestmagnitude of proliferation in PBMCs cultured in the absence of exogenouscytokines such as IL-2.

In some embodiments, the lymphoproliferative element can comprise anintracellular domain that is an interleukin or an interleukin receptorand that w

as a part of a Top Construct identified in Libraries 1A, 1.1A, 2B, 2.1B,3A, 3.1A, 3B, 3.1B, 4B, and/or 4.1B of Examples 17 and 18 (Tables 7 to17). In some embodiments, the lymphoproliferative element can comprisean intracellular domain that is a cytokine receptor and that was a partof a Top Construct identified in Libraries 1A, 1.1A, 2B, 2.1B, 3A, 3.1A,3B, 3.1B, 4B, and/or 4.1B of Examples 17 and 18 (Tables 7 to 17). Insome embodiments, the lymphoproliferative element can comprise anintracellular domain that includes at least one ITAM motif and that wasa part of a Top Construct identified in Libraries 1A, 1.1A, 2B, 2.1B,3A, 3.1A, 3B, 3.1B, 4B, and/or 4.1B of Examples 17 and 18 (Tables 7 to17).

In illustrative embodiments, the lymphoproliferative element cancomprise an intracellular domain from IL7R, IL12RB1, IL15RA, or IL27RA,which were present in constructs that showed particularly noteworthyenrichments (i.e. elicited highest magnitude of proliferation) in aninitial screen and a repeated screen as detailed in Examples 17 and 18(Tables 19 to 23).

In illustrative embodiments, the lymphoproliferative element cancomprise an intracellular domain from the cytokine receptors CD27, CD40,CRLF2, CSF2RA, CSF3R, EPOR, FCER1G, FCGR2A, FCGR2C, GHR, IFNAR1, IFNAR2,IFNGR2, IL1R1, IL1RL1, IL2RA, IL2RG, IL3RA, IL5RA, IL6R, IL7R, IL9R,IL10RB, IL1IRA, IL12RB1, IL13RA1, IL13RA2, IL15RA, IL17RB, IL18R1,IL18RAP, IL20RB, IL22RA1, IL27RA, IL31RA, LEPR, MPL, OSMR, PRLR,TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, or TNFRSF18, which were present inconstructs that showed particularly noteworthy enrichments (i.e.elicited highest magnitude of proliferation) in an initial screen and arepeated screen as detailed in Examples 17 and 18 (Tables 19 to 23).

In illustrative embodiments, the lymphoproliferative element comprisesan intracellular domain from CD3D, CD3E, CD3G, CD79A, CD79B, FCER1G,FCGR2A, or FCGR2C, which include at least one ITAM motif and werepresent in constructs that showed particularly noteworthy enrichments(i.e. elicited highest magnitude of proliferation) in an initial screenand a repeated screen as detailed in Examples 17 and 18 (Tables 19 to23).

In some embodiments, the lymphoproliferative element in this paragraph,which were shown in examples 17 and 18 to be active in constructs withonly a single intracellular domain, can be the intracellular domain of alymphoproliferative element with either two or more intracellulardomains, or in illustrative embodiments a single intracellular domain,i.e. the lymphoproliferative element does not comprise two or moreintracellular domains. In illustrative embodiments, the intracellulardomain in a lymphoproliferative element comprises a domain from CD40,CRLF2, CSF2RA, CSF3R, EPOR, FCGR2A, IFNAR2, IFNGR2, IL1R1, IL3RA, IL7R,IL10RB, IL11RA, IL12RB1, IL13RA2, IL18RAP, IL31RA, MPL, MYD88, TNFRSF14,or TNFRSF18, which were present in constructs that showed particularlynoteworthy enrichments (i.e. elicited highest magnitude ofproliferation) in an initial screen and a repeated screen as detailed inExample 18 as single intracellular signaling domains (Tables 22 and 23).In illustrative embodiments, the intracellular domain in alymphoproliferative element comprises a domain from IL7R or IL12RB1,which were present in constructs that showed particularly noteworthyenrichments (i.e. elicited highest magnitude of proliferation) in aninitial screen and a repeated screen as detailed in Example 18 (Tables22 and 23). In some embodiments, the intracellular domain in alymphoproliferative element with a single intracellular domain can be acytokine receptor. In illustrative embodiments, the cytokine receptor ina lymphoproliferative element with a single intracellular domaincomprises a domain from CD40, CRLF2, CSF2RA, CSF3R, EPOR, FCGR2A,IFNAR2, IFNGR2, IL1R1, IL3RA, IL7R, IL10RB, IL11RA, IL12RB1, IL13RA2,IL18RAP, IL31RA, MPL, TNFRSF14, or TNFRSF18, which were present inconstructs that showed particularly noteworthy enrichments (i.e.elicited highest magnitude of proliferation) in an initial screen and arepeated screen as detailed in Example 18 (Tables 22 and 23). In someembodiments, the intracellular domain in a lymphoproliferative elementcan include at least one ITAM motif. In illustrative embodiments, theintracellular domain in a lymphoproliferative element that includes atleast one ITAM motif comprises a domain from FCGR2A, which was presentin constructs that showed particularly noteworthy enrichments (i.e.elicited highest magnitude of proliferation) in an initial screen and arepeated screen as detailed in Example 18 (Table 22).

In illustrative embodiments, the lymphoproliferative element cancomprise a costimulatory domain from CD27, CD28, OX40 (also referred toas TNFRSF4), GITR (also referred to as TNFRSF18), or HVEM (also referredto as TNFRSF14), which were present in constructs that showedparticularly noteworthy enrichments (i.e. elicited highest magnitude ofproliferation) in an initial screen and a repeated screen as detailed inExamples 17 and 18 (Tables 19 to 23).

In some embodiments, the lymphoproliferative element can be one of theconstructs M024-S190-S047, M025-S050-S197, M036-S170-S047,M012-S045-S048, M049-S194-S064, M025-S190-S050, M025-S190-S05,E013-T041-S186-S051, E013-T028-S186-S051, E014-T015-S186-S051,E011-T016-S186-S050, E011-T073-S186-S050, or E013-T011-S186-S211, all ofwhich stimulated proliferation of resting lymphocytes after transductionas shown in Example 21 (FIGS. 35 and 36 ). In certain embodiments,lymphoproliferative elements comprise an extracellular domain fromCSF3R, IL3RA, ICOS, CRLF, CSF2RA, LIFR, or CD40; a first intracellulardomain from MyD88, CD40, or MPL, and/or a second intracellular domainfrom CD27 or MyD88.

In some embodiments, the lymphoproliferative element can be theconstruct E013-T041-S186-S051, which stimulated proliferation of restinglymphocytes after transduction with a replication incompetentrecombinant retroviral particle displaying UCHT1scFvFc-GPI as shown andanalyzed in Example 22 (FIGS. 38A and B). In other embodiments thelymphoproliferative element is IL7-IL7RA-IL2RB, as shown and analyzed inExample 22.

As detailed in Example 17, for Libraries 1A and 1.1A, constructs withdomains from the cytokine receptors CD27, IL1RL1, IL6R, IL31RA, TNFRSF4,or TNFRSF18, showed particularly noteworthy enrichments (i.e. elicitedhighest magnitude of proliferation) in both Libraries 1A and 1.1A (Table19).

As detailed in Example 17, for Libraries 2B and 2.1B, constructs withdomains from the cytokine receptors CD40, FCER1G, FCGR2C, IFNGR2, GHR,IL10RB, IL11RA, IL13RA2, IL17RB, IL22RA1, TNFRSF14, or TNFRSF9, showedparticularly noteworthy enrichments (i.e. elicited highest magnitude ofproliferation) in both Libraries 2B and 2.1B (Table 20).

As detailed in Example 18, for Libraries 3A and 3.1A, constructs withdomains from the cytokine receptors CD27, CD40, CSF2RA, FCGR2A, MPL,OSMR, TNFRSF4, or TNFRSF18 showed particularly noteworthy enrichments(i.e. elicited highest magnitude of proliferation) in both Libraries 3Aand 3.1A (Table 21).

As detailed in Example 18, for Libraries 3B and 3.1B, constructs withdomains from the cytokine receptors CD27, CD40, CRLF2, CSF2RA, CSF3R,EPOR, FCER1G, FCGR2A, FCGR2C, IFNAR1, IFNAR2, IFNGR2, ILIRL1, IL2RG,IL3RA, IL5RA, IL6R, IL7R, IL9R, IL10RB, IL1IRA, IL12RB1, IL13RA1,IL13RA2, IL15RA, IL18RAP, IL20RB, IL27RA, IL31RA, LEPR, MPL, OSMR, PRLR,TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, or TNFRSF18, showed particularlynoteworthy enrichments (i.e. elicited highest magnitude ofproliferation) in both Libraries 3B and 3.1B (Table 22).

As detailed in Example 18, for Libraries 4B and 4.1B, constructs withdomains from the cytokine receptors CD27, CD40, CRLF2, CSF2RA, CSF3R,EPOR, FCGR2A, FCGR2C, IFNAR2, IFNGR2, IL1R1, IL2RA, IL3RA, IL2RG, IL6R,IL7R, IL10RB, IL1IRA, IL31RA, IL13RA1, IL13RA2, IL18R1, MPL, OSMR,TNFRSF4, TNFRSF9, or TNFRSF18, showed particularly noteworthyenrichments (i.e. elicited highest magnitude of proliferation) in bothLibraries 4B and 4.1B (Table 23).

In certain illustrative embodiments, the lymphoproliferative elementcomprises an intracellular domain of CD40, MPL and IL2Rb, which aredemonstrated in the Examples herein to promote PBMC proliferation.

In some embodiments, the lymphoproliferative element can be other than acytokine receptor. In some embodiments, the lymphoproliferative elementother than a cytokine receptor can include an intracellular signalingdomain from CD2, CD3D, CD3G, CD3Z, CD4, CD8RA, CD8RB, CD28, CD79A,CD79B, FCER1G, FCGR2A, FCGR2C, or ICOS. Exemplary embodiments ofchimeric lymphoproliferative elements that include these recited genesare provided in Examples 17 and 18, and the tables cited therein.

In some embodiments, CLE is other than IL-15 tethered to the IL-2/IL-15receptor.

In some of the methods and compositions disclosed herein, expression ofthe lymphoproliferative element is induced by and can even dependent onbinding of a compound to a control element (as discussed elsewhereherein), which in non-limiting embodiments is a ribowsitch. In someembodiments, the lymphoproliferative element is expressed from apromoter active in a T cell and/or an NK cell. For methods andcompositions provided herein, a skilled artisan will recognize thatpromoters are known that are active in T cells and/or NK cells and canbe used to express a first engineered signaling polypeptide or a secondengineered signaling polypeptide, or any component thereof. Inillustrative embodiments, such a promoter is not active in a packagingcell line, such as the packaging lines disclosed herein. In someembodiments, the promoter is the EF1a promoter or the murine stem cellvirus (MSCV) promoter (Jones et al., Human Gene Therapy (2009) 20:630-40). In illustrative embodiments, the promoter is the T cellspecific CD3 zeta promoter.

In some embodiments, the lymphoproliferative element is microenvironmentrestricted. For example, the lymphoproliferative element can be amutated receptor that binds its respective cytokine differentially inaberrant versus physiological conditions. For example, an IL-7R that canbind IL7 more strongly in a tumor environment than in a normalphysiological environment can be used.

In some embodiments, the lymphoproliferative element is fused to arecognition or elimination domain. Such recognition or eliminationdomains are disclosed in more detail herein. Such fusion provides theadvantage, especially when a truncated or other mutatedlymphoproliferative element is used, of requiring less polynucleotidesin the retroviral genome. This is important in illustrative embodimentsprovided herein, because it helps to permit more nucleic acids encodingfunctional elements to be included in the retroviral genome and becauseit adds a mechanism by which cells expressing the lymphoproliferativeelement can be killed if their proliferation is no longer wanted or isdetrimental to an organism.

In some embodiments, a lymphoproliferative element, including a CLE,comprises an intracellular activating domain as disclosed hereinabove.In some illustrative embodiments a lymphoproliferative element is a CLEcomprising an intracellular activating domain comprising anITAM-containing domain, As such, the CLE can comprise an intracellularactivating domain having at least 80%, 90%, 95%, 98%, or 100% sequenceidentity to the CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCERIG,FCGR2A, FCGR2C. DAP10/CD28, or ZAP70 domains provided herein. In certainillustrative embodiments, the intracellular activating domain is anITAM-containing domain from CD3D, CD3G, CD3Z, CD79A, CD79B, FCER1G,FCGR2A, or FCGR2C. CLEs comprising these intracellular activatingdomains are demonstrated in Examples 17 and 18 herein, and associatedtables 18-23, as being effective at promoting proliferation of PBMCs exvivo in cultures in the absence of exogenous cytokines such as exogenousIL-2. In some embodiments, provided herein are CLEs comprising anintracellular domain from CD3D, CD3G, CD3Z, CD79A, FCER1G.

In some embodiments, one or more domains of a lymphoproliferativeelement is fused to a co-stimulatory domain and/or an intracellularactivating domain of a CAR. A chimeric lymphoproliferative element asdisclosed herein, is not a chimeric antigen receptor (CAR) because itdoes not comprise an ASTR. However, in some embodiments, one or moreintracellular domains of a lymphoproliferative element can be part ofthe same polypeptide as a CAR or can be fused and optionallyfunctionally connected to some components of CARs. For example, alymphoproliferative element can be fused to an antigen-specifictargeting region (ASTR) and activated by binding of the ASTR to itsantigen. In still other embodiments, an engineered signaling polypeptidecan include an ASTR, an intracellular activation domain (such as a CD3zeta signaling domain), a co-stimulatory domain, and alymphoproliferative domain. Further details regarding co-stimulatorydomains, intracellular activating domains, ASTRs and other CAR domains,are disclosed elsewhere herein.

In illustrative embodiments herein, a T cell and/or NK cell survivalelement is introduced into a resting T cell and/or resting NK cell,typically by transducing the resting T cell and/or resting NK cell witha replication incompetent recombinant retroviral particle whose genomeencodes the T cell and/or NK cell survival element as part of anengineered signaling polypeptide. In some embodiments, alymphoproliferative element is also a T cell and/or NK cell survivalelement. As discussed above, some of the lymphoproliferative elementsnot only promote proliferation, but they promote cell survival as well.In some embodiments, the T cell and/or NK survival motif is not alymphoproliferative element. In some embodiments, the T cell and/or NKcell survival motif can be a CD28 T cell survival motif or a CD137 cellsurvival motif. Such T cell survival motifs can be found on engineeredsignaling polypeptides that include an ASTR, such as an scFv. In anillustrative embodiment, the T cell survival motif is a CD28 T cellsurvival motif or a CD137 motif connected to an scFv through a CD8atransmembrane domain or a CD28 transmembrane domain. In certainembodiments, said intracellular signaling domain comprises a polypeptidesequence comprising an immunoreceptor tyrosine-based activation motif(ITAM). In a certain embodiment, said polypeptide sequence is a CD3ζsignaling domain.

In some embodiments, the lymphoproliferative element is not apolypeptide, but rather comprises an inhibitory RNA. In someembodiments, methods, uses, compositions, and products of processesaccording to any aspect herein include both a lymphoproliferativeelement comprising an inhibitory RNA and a lymphoproliferative elementthat is an engineered signaling polypeptide. In embodiments where alymphoproliferative element is an inhibitory RNA, that inhibitory RNAcan be a miRNA that stimulates the STAT5 pathway typically bypotentiating activation of STAT5 by degrading or causing down-regulationof a negative regulator in the SOCS pathway. In some embodiments, themiRNA is directed to mRNA encoding proteins that affect proliferationsuch as but not limited to ABCG1, SOCS1, TGFbR2, SMAD2, cCBL, and PD1.In illustrative embodiments, as exemplified herein, such inhibitory RNA(e.g. miRNAs) can be located in introns in packaging cells and/or areplication incompetent recombinant retroviral particle genome and/or aretroviral vector, typically with expression driven by a promoter thatis active in a T cell and/or NK cell. Not to be limited by theory,inclusion of introns in transcription units are believed to result inhigher expression and/or stability of transcripts. As such, the abilityto place miRNAs within introns of a retroviral genome adds to theteachings of the present disclosure that overcome challenges in theprior art of trying to get maximum activities into the size restrictionsof a retroviral, such as a lentivirus genome. In some embodiments, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 miRNAs, in illustrative embodiments between 2and 5, for example 4 miRNAs, one or more of which each bind nucleicacids encoding one or more of ABCG1, SOCS1, TGFbR2, SMAD2, cCBL, andPD1, can be included in the recombinant retroviral genome and deliveredto a target cell, for example T cells and/or NK cells, using methodsprovided herein. In fact, as provided herein 1, 2, 3, or 4 miRNAs can bedelivered in a single intron such as the EF1a intron.

ABCG1 is an ATP-binding cassette transporter that negatively regulatesthymocyte and peripheral lymphocyte proliferation (Armstrong et al.2010. J Immunol 184(1):173-183).

SOCS1 is a member of the SOCS (Suppressor of cytokine signaling) familyof negative regulators of cytokine signal transduction that inhibit theJak/Stat pathway such as STATS. SOCS1 is also known as JAB (Janus Kinasebinding protein), SSI-1 (Stat-induced Stat inhibitor-1), and TIP3(Tec-interacting protein).

TGFbR2 is a member of the serine/threonine protein kinase family thatbinds TGF-β, forming a complex that phosphorylates proteins that thenenter the nucleus and regulate transcription of genes related toproliferation.

SMAD2 mediates the signal of the transforming growth factor (TGF)-β andregulates multiple cellular processes, such as cell proliferation,apoptosis, and differentiation.

cCBL is an E3 ubiquitin ligase that inhibits TCR signaling bydephosphorylation and inactivation of ZAP-70 and through internalizationof the TCR.

PD1 (CD279) is a cell surface receptor expressed on T cells and ProBcells. PD-1 binds two ligands, PD-L1 and PD-L2. Signaling through PD-1functions to prevent activation of cells.

In some embodiments, herein the lymphoproliferative element is apolypeptide comprising the intracellular region of any of the genes ofTable 18. In some embodiments, the lymphoproliferative element comprisesor is an intracellular domain identified in the chimeric polypeptides inTable 18. In these embodiments, the lymphoproliferative element can be apolypeptide that is a chimeric polypeptide (See CLEs below), or is not aCLE but comprises or is an intracellular domain of a gene identified inthe P3 (first intracellular domain) position of Table 18. Table 18identifies CLE constructs that promoted cell proliferation of PBMCsbetween day 7 and the last day where a second intracellular domain wasnot present on the construct. In some subembodiments of this embodiment,the lymphoproliferative element is a chimeric polypeptide (i.e. aCLE—see below) that includes a combination of transmembrane domain andintracellular domain, with or without an extracellular domain comprisinga dimerizing motif.

In some embodiments, the lymphoproliferative element comprises MPL, oris MPL, or a variant and/or fragment thereof, including a variant and/orfragment that includes at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or100% of the intracellular domain of MPL, with or without a transmembraneand/or extracellular domain of MPL, and/or has at least 75, 80, 85, 90,95, 96, 97, 98, 99, or 100% sequence identity to the intracellulardomain of MPL, with or without a transmembrane and/or extracellulardomain of MPL, wherein the variant and/or fragment retains the abilityto promote cell proliferation of PBMCs, and in some embodiments T cells.In some embodiments, an MPL fragment included in the compositions andmethods herein has and/or retains a JAK-2 binding domain. In someembodiments, an MPL fragment included herein has or retains the abilityto activate a STAT. The full intracellular domain of MPL is SEQ IDNO:491 (part S186 in Tables 7 to 18). MPL is the receptor forthrombopoietin. Several cytokines such as thrombopoietin and EPO arereferred to in the literature and herein as either a hormone or acytokine.

In some embodiments, which provide separate aspects of the presentdisclosure, provided herein are chimeric polypeptides that are chimericlymphoproliferative elements (CLEs), as well as isolated polynucleotidesand nucleic acid sequences that encode the same. Exemplary CLEs areillustrated in FIGS. 30-33 . CLEs herein promote cell proliferation of Tcells and/or NK cells and can optionally also promote survival of Tcells and/or NK cells. Some CLEs promote proliferation and optionallyalso survival of other types of PBMCs, for example B cells. Embodimentsprovided herein that include nucleic acid sequences that encode a CLEand a CAR can be referred to herein as CLE CAR polynucleotideembodiments for drafting convenience. In some embodiments, CLEs caninclude a transmembrane domain and a first intracellular domain.Furthermore, in illustrative embodiments, as shown in FIGS. 30-33 , CLEsinclude an extracellular domain and/or a second intracellular domain(and in further embodiments, third, fourth, etc. intracellular domains).Chimeric polypeptides herein are chimeric because at least one domain isfrom a different polypeptide than at least one of the other domains andsuch chimera are not found naturally in an organism without humanintervention. Some CLEs include a ligand for a receptor and some CLEs donot include a ligand for a receptor.

Not to be limited by theory, such CLEs were designed to promoteproliferation and optionally cell survival in B cells, NK cells, and/orT cells in a constitutive manner (i.e. without the requirement of ligandbinding for activation). None of the CLEs are found in nature, and manyof the CLEs have components that are not usually expressed in B cells, Tcells, and/or NK cells in vivo and/or some candidate CLEs are notgenerally known to specifically promote cell proliferation and/or cellsurvival signaling in B cells, T cells, and/or NK cells. For example,MPL is usually not expressed in B cells, T cells and/or NK cells.Surprisingly, new CLEs were identified by screening a large number ofcandidate chimeric polypeptides as set out in Examples 17 and 18, thatpromoted PBMC cell proliferation. Such CLEs help to meet the long-feltneed of identifying mechanisms to stimulate proliferation and optionallysurvival as well, of B cells, T cells, and/or NK cells, such as would bebeneficial for important clinically-relevant technologies, such as CAR-Tand T-cells that are genetically engineered to express a defined TCR. Assuch, it is believed that some CLEs will provide a T cell and/or NK cellthe ability to expand in vivo without the need for lymphodepleting thehost.

Chimeric lymphoproliferative elements provided herein, and isolatedpolynucleotides and nucleic acids encoding the same can be included inany aspect provided herein that includes a lymphoproliferative element.For example, a first engineered signaling polypeptide can be, or caninclude a CLE in aspects provided herein that include one or moretranscriptional units that encode a first engineered signalingpolypeptide regulated by a control element. Furthermore, separateaspects of the invention are provided that specifically include a CLE.For example, such aspects include isolated chimeric lymphoproliferativepolypeptides, isolated polynucleotides and nucleic acid sequencesencoding the same, and vectors, including plasmids, viral, andretroviral vectors including such nucleic acid sequences or isolatedpolynucleotides. Such aspects further include, methods for transducingor transfecting PBMCs, such as B cells, or especially T cells and NKcells, with the isolated polynucleotides and vectors comprising thesame. Such cells can be isolated, unaltered cells, or they can be cellsthat have been modified such as cells that are genetically modified,such as to express a defined TCR, or CAR-T cells.

In some embodiments, CLEs include both an extracellular portion and atransmembrane portion that is from the same protein, in illustrativeembodiments the same receptor, either of which in illustrativeembodiments is a mutant, thus forming an extracellular and transmembranedomain. These domains can be from a cytokine receptor, or a mutantthereof, or a hormone receptor, or a mutant thereof in some embodimentsthat have been reported to be constitutively active when expressed atleast in some cell types. In illustrative embodiments, suchextracellular and transmembrane domains do not include a ligand bindingregion. It is believed that such domains do not bind a ligand whenpresent in CLEs and expressed in B cells, T cells, and/or NK cells.Mutations in such receptor mutants can occur in the transmembrane regionor in the extracellular juxtamembrane region. Not to be limited bytheory, a mutation in at least some extracellular—transmembrane domainsof CLEs provided herein, are responsible for signaling of the CLE in theabsence of ligand, by bringing activating chains together that are notnormally together, or by changing the confirmation of a linkedtransmembrane and/or intracellular domain.

One aspect that utilizes such transmembrane domains of receptor mutants,provided herein is an isolated polynucleotide comprising one or morenucleic acid sequences, wherein:

-   -   a first nucleic acid sequence of the one or more nucleic acid        sequences encodes a chimeric polypeptide comprising in amino to        carboxy orientation,    -   (a) an extracellular and transmembrane domain from a cytokine        receptor or a hormone receptor, wherein at least one of the        extracellular domain and the transmembrane domain comprise a        mutation that is found on a constitutively active mutant of the        cytokine receptor and wherein the extracellular sequence does        not bind a ligand of the cytokine receptor; and    -   (b) a first intracellular domain selected from an intracellular        domain of a gene having a first intracellular domain and        optionally a second domain of a selected polypeptide identified        in Tables 7 to 11, wherein said chimeric polypeptide promotes        cell proliferation of B cells, T cells, and/or NK cells.

The extracellular region of such extracellular and transmembrane domainsin these embodiments, is typically long enough to form a linker, inillustrative embodiments a flexible linker between a transmembranedomain and another functional peptide region, such as a clearancedomain, that in some embodiments, is linked to the amino terminus of theextracellular region. Accordingly, the extracellular region when presentto form an extracellular and transmembrane domain, can be between 1 and1000 amino acids, and is typically between 4 and 400 amino acids,between 4 and 200 amino acids, between 4 and 100 amino acids, between 4and 50 amino acids, between 4 and 25 or between 4 and 20 amino acids inlength. In one embodiment, the extracellular region is GGGS for anextracellular and transmembrane domain of this aspect of the invention.

The transmembrane domain whether as part of embodiments that include anextracellular and transmembrane domain as one part, for example as shownin Libraries 1A, 1.1A, 1.1B, 2B and 2.1B of Example 17, or as part ofembodiments that include an extracellular dimerizing motif, as shown inLibraries 3A, 3B, 3.1A, 3.1B, 4B, and 4.1B of Example 18, as discussedin more detail below, are typically at least long enough to cross aplasma membrane. Accordingly, transmembrane domains or transmembraneregions of extracellular and transmembrane domains, can be between 10and 250 amino acids, and are more typically at least 15 amino acids inlength, and can be for example between 15 and 100, 15 and 75, 15 and 50,15 and 40, or 15 and 30 amino acids.

Exemplary extracellular and transmembrane domains for CLEs ofembodiments that include such domains, in illustrative embodiments, areextracellular regions, typically less than 30 amino acids of themembrane-proximal extracellular domains along with transmembrane domainsfrom mutant receptors that have been reported to be constitutive, thatis not require ligand binding for activation of an associatedintracellular domain. In illustrative embodiments, such extracellulartransmembrane domains include IL7RA Ins PPCL, CRLF2 F232C, CSF2RB V449E,CSF3R T640N, EPOR L251C I252C, GHR E260C I270C, IL27RA F523C, and MPLS505N. Further non-limiting examples of such extracellular andtransmembrane domains are provided in Table 6 and exemplified in Example17 and the corresponding tables. In some embodiments, the extracellularand transmembrane domain does not comprise more than 10, 20, 25 30 or 50consecutive amino acids that are identical in sequence to a portion ofthe extracellular and/or transmembrane domain of IL7RA, or a mutantthereof. In some embodiments, the extracellular and transmembrane domainis other than IL7RA Ins PPCL. In some embodiments, the extracellular andtransmembrane does not comprise more than 10, 20, 25, 30, or 50consecutive amino acids that are identical in sequence to a portion ofthe extracellular and/or transmembrane domain of IL15R.

Additional exemplary transmembrane domains are provided in Example 18.These transmembrane domains in illustrative embodiments are from type Itransmembrane proteins.

Accordingly provided herein in one aspect is an isolated polynucleotidecomprising one or more nucleic acid sequences, wherein:

-   -   a first nucleic acid sequence of the one or more nucleic acid        sequences encodes a chimeric polypeptide comprising in amino to        carboxy orientation,        -   a) a transmembrane domain from a type I transmembrane            protein; and        -   b) a first intracellular domain selected from an            intracellular domain of a gene having a first intracellular            domain of a selected polypeptide identified in Tables 12 to            17, wherein said chimeric polypeptide promotes cell            proliferation of B cells, T cells, and/or NK cells.

In illustrative embodiments, said chimeric polypeptide promotes cellproliferation of PBMCs, for example B cells and/or NK cells, and/or inillustrative embodiments T cells. As shown in Example 18, such chimericpolypeptides are capable of promoting cell proliferation of PBMCs in theabsence of exposure of the PBMCs to exogenous cytokines such as IL-2,IL-15, or IL-7 during culturing, for example in the absence of addingIL-2 to culture media of PBMCs (e.g. B cells, T cells, or NK cells) thatexpress a nucleic acid encoding the chimeric polypeptide. Asillustrative in Example 18, for such embodiments IL-2 can be addedduring transduction of PBMCs, but not in subsequent culturing. Forexample, chimeric polypeptides disclosed herein are lymphoproliferativeelements because they are capable of promoting cell proliferation andoptionally cell survival of PBMCs, and in illustrative embodiments, Tcells, without adding IL-2 during culturing of PBMCs (e.g. T cells)after day 1, 2, 3, 4, 5, or 7 of culturing optionally after transductionof the PBMCs with a nucleic acid encoding the chimericlymphoproliferative element. Example 21 provides another example of amethod that can be used to identify and analyze lymphoproliferativeelements. Such analysis can include, for example, measuring the relativelymphoproliferative activity of such lymphoproliferative elements, forexample by analyzing the magnitude of enrichment provided by geneticallymodified lymphocytes expressing such lymphoproliferative elementscompared to control lymphocytes that do not express suchlymphoproliferative elements.

In one embodiment of this aspect, the first nucleic acid sequencefurther encodes an extracellular domain, and in illustrativeembodiments, the extracellular domain comprises a dimerizing motif. Inillustrative embodiments of this aspect, the extracellular domaincomprises a leucine zipper. In some embodiments, the leucine zipper isfrom a jun polypeptide, for example c-jun. In certain embodiments thec-jun polypeptide is the c-jun polypeptide region of ECD-11.

In embodiments of any of these aspects and embodiments wherein thetransmembrane domain is a type I transmembrane protein, thetransmembrane domain can be a Type I growth factor receptor, a hormonereceptor, a T cell receptor, or a TNF-family receptor. In an embodimentof any of the aspects and embodiments wherein the chimeric polypeptidecomprises an extracellular domain and wherein the extracellular domaincomprises a dimerizing motif, the transmembrane domain can be a Type Icytokine receptor, a hormone receptor, a T cell receptor, or aTNF-family receptor.

Exemplary transmembrane domains include any transmembrane domain thatwas used in Example 18. In some embodiments, the transmembrane domain isfrom CD4, CD8RB, CD40, CRLF2, CSF2RA, CSF3R, EPOR, FCGR2C, GHR, ICOS,IFNAR1, IFNGR1, IFNGR2, IL1R1, ILIRAP, IL2RG, IL3RA, IL5RA, IL6ST,IL7RA, IL10RB, IL1IRA, IL13RA2, IL17RA, IL17RB, IL17RC, IL17RE, IL18R1,IL18RAP, IL20RA, IL22RA1, IL31RA, LEPR, PRLR, and TNFRSF8, or mutantsthereof that are known to promote signaling activity in certain celltypes if such mutants are present in the constructs provided in Example18. In some embodiments, the transmembrane domain is from CD40, ICOS,FCGR2C, PRLR, IL3RA, or IL6ST. In some embodiments, the transmembrane isthe specific transmembrane domain provided for these genes in theconstructs of any one or more of Tables 12 to 17, or a transmembranedomain from any of the constructs in the first 50, 25 or 10 listedconstructs of those tables. In illustrative embodiments, thetransmembrane domain is from CD40 or ICOS, as non-limiting examples thetransmembrane domains for these genes provided in Table 6, or a fragmentthereof that retains the ability to be transmembrane, and/or mutantsthereof that are known to promote constitutive signaling activity incertain cell types.

Exemplary transmembrane domains from this aspect are shown as P2 inTables 12 to 17. For Library 3A, transmembrane domains (P2) from CD40,CD8B, CRLF2, CSF2RA, GCGR2C, ICOS, IFNAR1, IFNGR1, IL10RB, IL18R1,IL18RAP, IL3RA, LEPR, and PRLR, or mutants thereof that are known topromote constitutive signaling activity in certain cell types, if suchmutants are present in the constructs provided in Example 18, when foundat the transmembrane domain position (P2) of candidate chimericpolypeptides elements herein, promoted proliferation of PBMCs betweenday 7 and the last day, when data is considered in a combined manner forall constructs with a transmembrane domain derived from that gene. Inthe screens provided in Examples 17 and 18, cells were transduced withreplication incompetent recombinant retroviral particles containing alibrary and then either fed PBMCs, or not fed PBMCs, as discussed inExamples 17 and 18. Screens where the cells were fed PBMCs areidentified with an “A” after the library number, e.g. Library 1A andscreens where the cells were not fed PBMCs are identified with a “B”after the library number, e.g. Library 1.1B. Additionally, screensidentified as libraries containing “0.1”, e.g. Library 1.1A, wereperformed in an identical manner to screens of the corresponding librarywithout the “0.1” e.g. Library 1.1A was a repeat screen of Library 1A.For Library 3B, transmembrane domains from CD40, ICOS, CSF2RA, IL18R1,IL3RA, and TNFRSF14, or mutants thereof that are known to promoteconstitutive signaling activity in certain cell types, if such mutantsare present in the constructs provided in Example 18, were most capableof promoting cell proliferation. For Library 4B, transmembrane domainsfrom IL3RA, CSFR2RA, IL18R1, CD8B, CD40. ICOS, or mutants thereof thatare known to promote constitutive signaling activity in certain celltypes, if such mutants are present in the constructs provided in Example18, were most capable of promoting cell proliferation. In illustrativeembodiments, a transmembrane domain in a CLE provided herein is atransmembrane domain from CD40 and ICOS. Examples of transmembranedomains or portions and/or mutants thereof, that were present inconstructs that promoted cell proliferation for Libraries 3A, 3B, 3, 1A,3, 1B, 4B, and 4.1B are provided in the tables provided in Example 18.

In some embodiments of any aspect herein a chimeric polypeptidecomprises a transmembrane domain identified in any of the constructsshown in Tables 12 to 17 other than a transmembrane domain mutant V449Eof CSF2RB.

In some embodiments, the extracellular and transmembrane domain is theviral protein LMP1, or a mutant and/or fragment thereof. LMP1 is amultispan transmembrane protein that is known to activate cell signalingindependent of ligand when targeted to lipid rafts or when fused to CD40(Kaykas et al. EMBO J. 20: 2641 (2001)). A fragment of LMP1 is typicallylong enough to span a plasma membrane and to activate a linkedintracellular domain(s). For example, the LMP1 can be between 15 and386, 15 and 200, 15 and 150, 15 and 100, 18 and 50, 18 and 50, 18 and30, 20 and 200, 20 and 150, 20 and 50, 20 and 30, 20 and 100, 20 and 40,or 20 and 25 amino acids. A mutant and/or fragment of LMP1 when includedin a CLE provided herein, retains its ability to activate anintracellular domain. Furthermore, if present, the extracellular domainincludes at least 1, but typically at least 4 amino acids and istypically linked to another functional polypeptide, such as a clearancedomain, for example, an eTag.

In other embodiments of CLEs provided herein, the extracellular domainincludes a dimerizing moiety. Many different dimerizing moietiesdisclosed herein can be used for these embodiments. In illustrativeembodiments, the dimerizing moieties are capable of homodimerizing. Notto be limited by theory, dimerizing moieties can provide an activatingfunction on intracellular domains connected thereto via transmembranedomains. Such activation can be provided, for example, upon dimerizationof a dimerizing moiety, which can cause a change in orientation ofintracellular domains connected thereto via a transmembrane domain, orwhich can cause intracellular domains to come into proximity. Anextracellular domain with a dimerizing moiety can also serve a functionof connecting a recognition tag to a cell expressing a CLE. In someembodiments, the dimerizing agent can be located intracellularly ratherthan extracellularly. In some embodiments, more than one or multiples ofdimerizing domains can be used.

Extracellular domains for embodiments where extracellular domains have adimerizing motif, are long enough to form dimers, such as leucine zipperdimers. As such, extracellular domains that include a dimerizing moietycan be from 15 to 100, 20 to 50, 30 to 45, or 35 to 40 amino acids, ofin illustrative embodiments is a c-Jun portion of a c-Jun extracellulardomain provided in Table 6. Extracellular domains of polypeptides thatinclude a dimerizing moiety, may not retain other functionalities. Forexample, for leucine zippers embodiments, such leucine zippers arecapable of forming dimers because they retain a motif of leucines spaced7 residues apart along an alpha helix. However, leucine zipper moietiesof certain embodiments of CLEs provided herein, may or may not retaintheir DNA binding function.

One exemplary extracellular domain of this type, is a leucine zipperdomain from a Jun protein, such as c-Jun. For example, an extracellulardomain can be a variant of c-Jun found in NM_002228_3 (Table 6). Such anextracellular domain was used in the constructs discussed in Example 18.

A spacer of between 1 and 4 alanine residues can be included in CLEsbetween the extracellular domain that has a dimerizing moiety, and thetransmembrane domain. Not to be limited by theory, it is believed thatthe alanine spacer affects signaling of intracellular domains connectedto the leucine zipper extracellular region via the transmembrane domain,by changing the orientation of the intracellular domains.

The first and second intracellular domains of CLEs provided herein, areintracellular signaling domains of genes that are known in at least somecell types, to promote proliferation, survival (anti-apoptotic), and/orprovide a co-stimulatory signal that enhances a differentiation state,proliferative potential or resistance to cell death. As such, theseintracellular domains can be intracellular domains fromlymphoproliferative elements and co-stimulatory domains provided herein.Some of the intracellular domains of candidate chimeric polypeptides areknown to activate JAK1/JAK2 signaling and STAT5. The genes andintracellular domains thereof that are found in a first intracellulardomain are the same as the second intracellular domain, except that ifthe first and second intracellular domain are identical, then at leastone, and typically both the transmembrane domain and the extracellulardomain are not from the same gene.

Exemplary intracellular domains include those from the constructs listedin Tables 7 to 11. Exemplary intracellular domains from these genes thatwere empirically determined to be active in the experiment of Example17, are provided in the first intracellular domain (P3) and secondintracellular domain (P4) locations of tables provided in Example 17 andas further listed in this section below. Illustrative intracellulardomains identified in the top hits for the Example 17 screen includedCD40, CSF2RA, IFNAR1, ILIRAP, IL4R, IL6ST, IL1IRA, IL12RB2, IL17RA,IL17RD, IL17RE, IL18R1, IL21R, IL23R, MPL, and MyD88. Illustrativeintracellular domains identified in the top hits for the Example 18screen included CD40, LEPR, MyD88, IFNAR2, MPL, IL18R1, IL13RA2, IL10RB,IL23R, or CSF2RA. In certain illustrative embodiments, the firstintracellular domain is MPL, LEPR, MyD88, or IFNAR2. For these certainillustrative embodiments non-limiting exemplary second intracellulardomain (P4) domains are those of genes that are linked to thecorresponding MPL, LEPR, MyD88, IFNAR2, CD40, CD79B, or CD27 firstintracellular domain (P3) Tables 12 to 17, including in somenon-limiting examples the first and/or second intracellular domains forthose genes provided in these tables. For these certain illustrativeembodiments other non-limiting exemplary second intracellular (P4)domains are those of genes that are linked to the corresponding MPL,LEPR, MYD88, IFNAR2, CD40, CD79B, or CD27 first intracellular domain intables provided in Example 18, including as non-limiting examples, thefirst and/or second intracellular domains of those genes provided inthese tables. In some embodiments, the chimeric lymphoproliferativeelement can be any of the polypeptides in Tables 19-23 of Examples 17and 18.

In certain illustrative embodiments, the second intracellular domain isfrom CD3D, CD3G, CD27, CD40, CD79A, CD79B, FCER1G, FCGRA2, ICOS,TNFRSF4, and TNFRSF8, or mutants thereof that are known to promotesignaling activity in certain cell types if such mutants are present inthe constructs provided in Example 18, In certain illustrativeembodiments, the second intracellular domain is CD40, CD79B, TNFRSF4,TNFRSF9, TNFRSF14, FCGRA2, CD3G, or CD27, including in illustrativeexamples an intracellular domain of CD40, CD79B, and CD27. For thesecertain illustrative embodiments non-limiting exemplary firstintracellular domains (P3) are those of genes that are linked to CD40,CD79B, TNFRSF4, TNFRSF9, TNFRSF14, FCGRA2, CD3G, or CD27, including inillustrative examples an intracellular domain of CD40, CD79B, or CD27 inTables 12 to 17, including as non-limiting examples, the first and/orsecond intracellular domains of those genes provided in these tables.For these certain illustrative embodiments, other non-limiting exemplaryfirst intracellular (P3) domains are those of genes that are linked toCD40, CD79B, TNFRSF4, TNFRSF9, TNFRSF14, FCGRA2, CD3G, or CD27,including in illustrative examples an intracellular domain of CD40,CD79B, and CD27 second intracellular domains in tables for these genesas P3 provided in Example 18, including as non-limiting examples, thefirst and/or second intracellular domains of those genes provided inthese tables.

Detailed information, including sequence information, about exemplaryintracellular domains of the above genes that were identified inExamples 17 and 18 within constructs that promoted cell survival andproliferation are provided in Table 6. Intracellular domains that can beincluded in CLE embodiments provided herein include mutants and/orfragments of the intracellular domains of the recited genes providedthat such mutants and/or fragments retain the ability to promoteproliferation, survival (anti-apoptotic), and/or provide aco-stimulatory signal that enhances a differentiation state,proliferative potential or resistance to cell death. As such, theintracellular domains can be, for example, between 10 and 1000, 10 and750, 10 and 500, 10 and 250, 10 and 100 amino acids.

In some embodiments, all domains of a CLE are other than an IL-7receptor, or a mutant thereof, and/or a fragment thereof that has atleast 10, 15, 20, or 25 contiguous amino acids of IL-7 receptor, orother than an IL-15 receptor, or a mutant thereof, and/or a fragmentthereof that has at least 10, 15, 20, or 25 contiguous amino acids ofIL-15 receptor. In some embodiments, a CLE does not comprise acombination of first intracellular domain and second intracellulardomain of CD40 and MyD88.

In illustrative embodiments, CLEs include a recognition and/orelimination domain. Details regarding recognition and/or eliminationdomains are provided in other sections herein. Any of the recognitionand/or elimination domains provided herein can be part of a CLE.Typically the recognition domain is linked to the N terminus of theextracellular domain. Not to be limited by theory, in some embodiments,the extracellular domain includes the function of providing a linker, inillustrative embodiments a flexible linker, linking a recognition domainto a cell that expresses the CLE.

In illustrative embodiments, as illustrated in FIG. 30 and FIG. 32 , aCLE provided herein is co-expressed with a CAR which can be designedaccording to any of the CAR embodiments provided herein, or otherwiseknown in the art.

Some embodiments provided herein, are isolated polynucleotides andnucleic acid sequences encoding any of the CLEs provided herein, asexemplified in FIGS. 30-33 . Such isolated polynucleotides can includeany elements known in the art for providing expression of a transcript,such as a transcript encoding a CLE. For example, the isolatedpolynucleotide can include a promoter element that is active in B cells,T cells, and/or NK cells. Such promoters that are appropriate for theseembodiments are known in the art, some of which are identified in othersections of this disclosure. A skilled artisan will understand asdiscussed in more detail in Examples 17 and 18, for example, how todesign isolated polynucleotides and vectors containing the same, forexpressing any of the CLE embodiments provided herein. For example, insome embodiments, a Kozak sequence is provided within 10 nucleotidesupstream of an ATG start site encoding the amino terminal amine acid ofa CLE or a CAR located upstream a CLE on the same transcription unit.Accordingly, in polynucleotide embodiments comprising a nucleic acidencoding a CLE provided herein, the polynucleotide can further compriseone or more of a Kozak-related sequence, a WPRE element, and a multiplestop sequence, for example a double stop sequence or a triple stopsequence.

Furthermore, polynucleotides that include a nucleic acid sequenceencoding a CLE provided herein, also typically comprise a signalsequence to direct expression to the plasma membrane. Exemplary signalsequences are provided herein in other sections. Elements can beprovided on the transcript such that both a CAR and CLE are expressedfrom the same transcript. Such a construct is advantageous in requiringless nucleic acids than if these elements were expressed from differenttranscripts, which is an important feature that allows such constructsto be present in vectors, such as retroviral genomes, used to transfector transduce B cells, T cells, and/or NK cells, for example.

Numerous chimeric polypeptide candidates were designed and identified aslymphoproliferative elements in Examples 17 and 18 herein. For Library1A, which included constructs that encoded the chimeric polypeptides anda CAR, 172 top candidate chimeric polypeptides were identified (SeeTable 7) that promoted PBMC proliferation between day 7 and the last daywhen cultured in the absence of IL-2. For Library 2B, which includedconstructs that encoded the chimeric polypeptides but did not include aCAR, 167 top candidate chimeric polypeptides were identified (See Table8) that promoted PBMC proliferation between day 7 and the last day whencultured in the absence of IL-2. Certain illustrative CLEs, as well aspolynucleotides, and nucleic acid sequences encoding the same, andmethods that include any of these, include intracellular domains fromgenes having matched domains (e.g. transmembrane gene and firstintracellular gene and optionally second intracellular gene) onconstructs provided in Tables 7 to 11, as well as, in non-limitingexamples, the specific matched domains provided on these tables, some ofwhich are set out in the Exemplary Embodiments section herein. Inillustrative embodiments, the intracellular domains from CLEs havingmatched domains (e.g. transmembrane gene and first intracellular geneand optionally second intracellular gene) on constructs that hadparticularly noteworthy enrichments in a first screen and a repeatedscreen with the same P1-P2, P3 and P4 domains, e.g. Libraries 1A and1.1A, can be used, as shown in Tables 7 to 11. For Libraries 1A and1.1A, the constructs M001-S116-S044, M024-S192-S045, M001-S047-S102,M048-S195-S043, M012-S216-S211, and M030-S170-S194 had particularlynoteworthy enrichments in both screens.

Further information about the first and second intracellular domains inconstructs with particularly noteworthy enrichments in screens of bothLibrary 1A and Library 1.1A is provided in Table 19, including thegene(s) the first and second intracellular domain are derived from,whether the first and/or second intracellular domain are cytokinereceptors, and whether the first and/or second intracellular domain haveat least one ITAM motif. As shown in Example 17, constructs withintracellular domains from IL6R, MYD88, CD27, MYD88, TNFRSF18, orIL31RA, when present at the first intracellular domain (P3), andconstructs with intracellular domains from CD8B, IL1RL1, CD8A, TNFRSF4,or MYD88, when present at the second intracellular domain (P4), showedparticularly noteworthy enrichments in both Libraries 1A and 1.1A (Table19). Constructs with domains from the cytokine receptors IL6R, CD27,TNFRSF18, or IL31RA, when present at the first intracellular domain(P3), and constructs with domains from the cytokine receptors IL1RL1 orTNFRSF4, when present at the second intracellular domain (P4), showedparticularly noteworthy enrichments in both Libraries 1A and 1.1A (Table19).

For Libraries 2B and 2.1B, the constructs M007-S049-S051,M007-S050-S039, M012-S050-S043, M012-S161-S213, M030-S142-S049,M001-S145-S130, M018-S085-S039, M018-S075-S053, M012-S135-S074, andM007-S214-S077 had particularly noteworthy enrichments in both screens.For the purposes of the repeated screens, constructs with particularlynoteworthy enrichments were those that had a log₂ ((normalized countdata on the last day+1)/(normalized count data on day 7+1)) value above2.

Further information about the first and second intracellular domains inconstructs with particularly noteworthy enrichments in screens of bothLibrary 2B and Library 2.1B is provided in Table 20, including thegene(s) the first and second intracellular domain are derived from,whether the first and/or second intracellular domain are cytokinereceptors, and whether the first and/or second intracellular domain haveat least one ITAM motif. Constructs with intracellular domains fromCD28, CD40, IL22RA1, IL13RA2, IL17RB, IFNGR2, FCGR2C, IL11RA, orTNFRSF14, when present at the first intracellular domain (P3), andconstructs with intracellular domains from CD40, CD3G, CD8A, TNFRSF9,CD28, IL10RB, CD79B, FCER1G, or GHR, when present at the secondintracellular domain (P4), showed particularly noteworthy enrichments inboth Libraries 2B and 2.1B (Table 20). Constructs with domains from thecytokine receptors CD40, IL22RA1, IL13RA2, IL17RB, IFNGR2, FCGR2C,IL11RA, or TNFRSF14, when present at the first intracellular domain(P3), and constructs with domains from the cytokine receptors TNFRSF9,IL10RB, FCER1G, GHR, or CD40, when present at the second intracellulardomain (P4), showed particularly noteworthy enrichments in bothLibraries 2B and 2.1B (Table 20). Constructs with domains containingITAM motifs from FCGR2C, when present at the first intracellular domain(P3), and constructs with domains containing ITAM motifs from CD3G,CD79B, or FCER1G, when present at the second intracellular domain (P4),showed particularly noteworthy enrichments in both Libraries 2B and 2.1B(Table 20).

In an initial interim analysis, for Library 3A, which includedconstructs that encoded the chimeric polypeptides and a CAR, 126 topcandidate chimeric polypeptides were identified (See Table 12) thatpromoted PBMC proliferation between day 7 and the last day in transducedPBMCs stimulated with untransduced PBMCs as set out in Example 18, whencultured in the absence of IL-2. For the initial interim analysis ofLibrary 3B, which included constructs that encoded the chimericpolypeptides and a CAR, 127 top candidate chimeric polypeptides wereidentified (See Table 13) that promoted PBMC proliferation between day 7and the last day in transduced PBMCs that were not stimulated withuntransduced PBMCs as set out in Example 18, when cultured in theabsence of IL-2. For Library 4B, which included constructs that encodedthe chimeric polypeptides but not a CAR, 154 top candidate chimericpolypeptides were identified (See Table 16) that promoted PBMCproliferation between day 7 and the last day in transduced PBMCs thatwere not stimulated with untransduced PBMCs as set out in Example 18,when cultured in the absence of IL-2.

After further decoding, which provided deep decoding, in Libraries 3Aand 3B, which included constructs that encoded the chimeric polypeptidesand a CAR and transduced PBMCs that were supplemented with (Library 3A)or without (Library 3B) fresh untransduced PBMCs, 134 top candidateswere identified for Library 3A at day 21, 124 top candidates wereidentified for Library 3A at day 35, and 131 top candidates wereidentified for Library 3B at day 21 (See Tables 12 and 13, respectively)that promoted PBMC proliferation between day 7 and the last day whencultured the absence of IL-2 (i.e. IL-2 was not added to the culturemedium during culturing after the initial transduction), IL-7, or anyother exogenous cytokine.

Certain illustrative CLEs, as well as polynucleotides, and nucleic acidsequences encoding the same, and methods that include any of these,include intracellular domains from genes having matched domains (e.g.transmembrane gene and first intracellular gene and optionally secondintracellular gene) on constructs provided in Tables 12 to 17, ormutants thereof that retain signaling activity, as well as, innon-limiting examples, the specific matched domains provided on thesetables, some of which are set out in the Exemplary Embodiments sectionherein. Intracellular domains that are identified in the data providedin Example 17 and Example 18, from either the first intracellular domainor the second domain position, are envisioned in exemplary CLEembodiments, interchangeably as either the first or second intracellulardomain. In illustrative embodiments, the intracellular domains fromgenes having matched domains (e.g. transmembrane gene and firstintracellular gene and optionally second intracellular gene) onconstructs that had particularly noteworthy enrichments in a firstscreen and a repeated screen with the same P1, P2, P3, and P4 domains,e.g. Libraries 3A and 3.1A, can be used. For Libraries 3A and 3.1A, theconstructs E008/E013-T041-S186-S050, E006/E011-T077-S186-S211,E007/E012-T021-S186-S051, E009/E014-T041-S186-S053,E007/E012-T073-S186-S053, E006/E011-T017-S186-S051,E006/E011-T031-S186-S211, E006/E011-T011-S186-S050,E006/E011-T011-S186-S047, E007/E012-T001-S186-S050,E006/E011-T041-S186-S051, E008/E013-T028-S186-S076,E009/E014-T029-S199-S053, E009/E014-T062-S186-S216,E007/E012-T006-S058-S051, E009/E014-T076-S186-S211, andE007/E012-T001-S186-S047 had particularly noteworthy enrichments in bothscreens, where the P1 parts separated by a slash here included differenttags for Libraries 3A and 3A.1 as shown in Tables 6, 12, and 14. For thepurposes of the repeated screens, constructs with particularlynoteworthy enrichments were those that had a log₂ ((normalized countdata on the last day+1)/(normalized count data on day 7+1)) value above2.

Further information about the first and second intracellular domains inconstructs with particularly noteworthy enrichments in screens of bothLibrary 3A and Library 3.1A is provided in Table 21, including thegene(s) the first and second intracellular domain are derived from,whether the first and/or second intracellular domain are cytokinereceptors, and whether the first and/or second intracellular domain haveat least one ITAM motif. Constructs with intracellular domains from MPL,OSMR, or CSF2RA, when present at the first intracellular domain (P3),and constructs with intracellular domains from CD40, TNFRSF4, CD79B,CD27, FCGR2A, or TNFRSF18, when present at the second intracellulardomain (P4), showed particularly noteworthy enrichments in bothLibraries 3A and 3.1A (Table 21). Constructs with domains from thecytokine receptors MPL, OSMR, or CSF2RA, when present at the firstintracellular domain (P3), and constructs with domains from the cytokinereceptors CD40, TNFRSF4, CD27, FCGR2A, or TNFRSF18, when present at thesecond intracellular domain (P4), showed particularly noteworthyenrichments in both Libraries 3A and 3.1A (Table 21). Constructs withdomains containing ITAM motifs from MPL or OSMR, when present at thesecond intracellular domain (P4), showed particularly noteworthyenrichments in both Libraries 3A and 3.1A (Table 21).

For Libraries 3B and 3.1B, the constructs E007/E012-T017-S186-S051,E007/E012-T073-S186-S053, E008/E013-T028-S186-S047,E006/E011-T011-S186-S047, E007/E012-T082-S176-S214,E006/E011-T046-S186-S052, E008/E013-T029-S186-S052,E009/E014-T011-S186-S053, E008/E013-T032-S186-S039,E007/E012-T034-S186-S051, E007/E012-T041-S192-S213,E006/E011-T014-S069-5213, E006/E011-T022-S186-S053,E006/E011-T023-S115-S075, E006/E011-T029-S106-S213,E006/E011-T032-S155-S080, E006/E011-T041-S186-S216,E006/E011-T057-S135-S080, E006/E011-T072-S191-X002,E006/E011-T077-S186-S216, E006/E011-T080-S141-S080,E007/E012-T001-X001-5214, E007/E012-T007-S059-S211,E007/E012-T016-S186-S052, E007/E012-T031-S186-S053,E007/E012-T044-S102-S052, E007/E012-T044-S142-X002,E007/E012-T055-S069-S053, E007/E012-T063-S176-S216,E007/E012-T065-S157-S075, E008/E013-T008-S085-X002,E008/E013-T011-S085-S048, E008/E013-T021-S109-X002,E008/E013-T021-S168-S211, E008/E013-T032-S064-S214,E008/E013-T037-S170-S215, E008/E013-T038-S176-S048,E008/E013-T039-S137-S216, E008/E013-T041-S141-S053,E008/E013-T045-S177-S048, E008/E013-T048-S109-S074,E008/E013-T073-S199-S075, E009/E014-T001-S157-S074,E009/E014-T005-S196-S049, E009/E014-T011-S130-X002,E009/E014-T013-S155-X002, E009/E014-T017-S186-S076,E009/E014-T021-S142-S080, E009/E014-T023-S082-S076,E009/E014-T038-S196-S037, E009/E014-T055-S186-S052,E009/E014-T060-S175-S053, E009/E014-T070-S085-S212,E008/E013-T026-S054-S213, E009/E014-T007-S120-S053,E007/E012-T045-S186-S211, E008/E013-T073-S186-X002,E008/E013-T074-S186-X002, E007/E012-T055-S186-S053,E008/E013-T036-S186-S053, E007/E012-T017-S058-S053,E008/E013-T030-S189-5080, E006/E011-T029-S081-S047,E009/E014-T044-S194-S050, E006/E011-T028-S121-X002,E008/E013-T028-S186-S053, E009/E014-T078-S142-S213,E009/E014-T041-S186-S051, E008/E013-T006-S186-S050,E006/E011-T028-S186-S075, E006/E011-T040-S120-S038,E007/E012-T044-S115-S211, E009/E014-T039-S176-S075,E007/E012-T028-S186-S050, E008/E013-T031-S202-S050,E007/E012-T072-S192-S053, E006/E011-T065-X001-S051,E007/E012-T030-S062-X002, E007/E012-T073-S186-X002,E009/E014-T056-S186-S053, E008/E013-T046-S137-X002,E006/E011-T016-S136-5076, E007/E012-T032-S142-S037,E007/E012-T065-S120-S215, E009/E014-T077-S186-S047,E009/E014-T001-S126-S051, E006/E011-T030-S121-S039,E008/E013-T006-S176-S213, E009/E014-T032-S130-S215,E008/E013-T041-S186-S039, E009/E014-T021-S186-S047,E008/E013-T026-S137-S214, E007/E012-T029-S116-S075,E008/E013-T026-S106-S049, and E007/E012-T032-S168-S075 had particularlynoteworthy enrichments in both screens, where the P1 parts separated bya slash here included different tags for Libraries 3B and 3.1B as shownin Tables 6, 13, and 15. For the purposes of the repeated screens,constructs with particularly noteworthy enrichments were those that hada log₂ ((normalized count data on the last day+1)/(normalized count dataon day 7+1)) value above 2.

Further information about the first and second intracellular domains inconstructs with particularly noteworthy enrichments in screens of bothLibrary 3B and Library 3.1B is provided in Table 22, including thegene(s) the first and second intracellular domain are derived from,whether the first and/or second intracellular domain are cytokinereceptors, and whether the first and/or second intracellular domain haveat least one ITAM motif. Constructs with intracellular domains from MPL,LEPR, MYD88, EPOR, IL5RA, IL2RG, IL18RAP, IL1IRA, IL13RA1, CSF2RA,IL1RL1, IL13RA2, IL20RB, IFNGR2, IL3RA, IL27RA, CSF3R, IL31RA, IL12RB1,OSMR, IL10RB, IFNAR2, CRLF2, IL7R, IFNAR1, PRLR, IL9R, IL6R, or IL15RA,when present at the first intracellular domain (P3), and constructs withintracellular domains from CD40, CD79B, CD27, TNFRSF14, CD79A, CD3G,TNFRSF9, FCGR2C, ICOS, TNFRSF18, TNFRSF4, CD28, FCER1G, FCGR2A, CD3D,TNFRSF8, or CD3E, when present at the second intracellular domain (P4),showed particularly noteworthy enrichments in both Libraries 3B and 3.1B(Table 22). Constructs with domains from the cytokine receptors MPL,LEPR, EPOR, IL5RA, IL2RG, IL18RAP, IL1IRA, IL13RA1, CSF2RA, ILIRL1,IL13RA2, IL20RB, IFNGR2, IL3RA, IL27RA, CSF3R, IL31RA, IL12RB1, OSMR,IL10RB, IFNAR2, CRLF2, IL7R, IFNAR1, PRLR, IL9R, IL6R, or IL15RA, whenpresent at the first intracellular domain (P3), and constructs withdomains from the cytokine receptors CD40, CD27, TNFRSF14, TNFRSF9,FCGR2C, TNFRSF18, TNFRSF4, FCER1G, FCGR2A, or TNFRSF8, when present atthe second intracellular domain (P4), showed particularly noteworthyenrichments in both Libraries 3B and 3.1B (Table 22). Constructs withdomains containing ITAM motifs from CD79B, CD79A, CD3G, FCGR2C, FCER1G,FCGR2A, CD3D, or CD3E, when present at the second intracellular domain(P4), showed particularly noteworthy enrichments in both Libraries 3Band 3.1B (Table 22).

For Libraries 4B and 4.1B, the constructs E007/E012-T078-S154-S047,E008/E013-T062-S186-X002, E008/E013-T055-S186-S050,E009/E014-T057-S186-S050, E007/E012-T077-S054-S053,E007/E012-T034-S135-S211, E009/E014-T071-X001-S216,E009/E014-T011-S141-S037, E008/E013-T041-S186-S037,E006/E011-T038-S106-S039, E006/E011-T011-S121-X002,E007/E012-T007-S085-5215, E006/E011-T041-S186-S050,E007/E012-T008-S064-S051, E006/E011-T041-S186-S047,E008/E013-T045-S186-S051, E008/E013-T003-S104-S216,E006/E011-T019-S186-S053, E008/E013-T071-S064-S080,E006/E011-T021-S054-X002, E006/E011-T003-S135-X002,E009/E014-T020-S199-S213, E008/E013-T027-S121-S211,E009/E014-T032-S195-X002, E009/E014-T050-S171-X002,E008/E013-T069-S083-X002, E008/E013-T026-S116-S038,E009/E014-T072-S195-X002, E007/E012-T047-S058-S211,E008/E013-T046-S142-S080, E006/E011-T065-S186-S076,E006/E011-T062-S069-X002, E007/E012-T047-S098-X002,E009/E014-T069-S099-S048, E008/E013-T039-S141-S050,E006/E011-T052-S130-S052, E008/E013-T041-S186-X002,E007/E012-T019-S120-X002, E008/E013-T045-S186-S053,E006/E011-T003-S170-S039, E007/E012-T047-S058-S051,E007/E012-T069-S109-X002, E009/E014-T019-S130-S075, andE006/E011-T047-S054-S053 had particularly noteworthy enrichments in bothscreens, where the P1 parts separated by a slash here included differenttags for Libraries 4B and 4.1B as shown in Tables 6, 16, and 17. For thepurposes of the repeated screens, constructs with particularlynoteworthy enrichments were those that had a log₂ ((normalized countdata on the last day+1)/(normalized count data on day 7+1)) value above2. Further information about the first and second intracellular domainsin constructs with particularly noteworthy enrichments in screens ofboth Library 4B and Library 4.1B is provided in Table 23, including thegene(s) the first and second intracellular domain are derived from,whether the first and/or second intracellular domain are cytokinereceptors, and whether the first and/or second intracellular domain haveat least one ITAM motif. Constructs with intracellular domains fromIL18R1, MPL, CRLF2, IL11RA, IL13RA1, IL2RG, IL7R, IFNGR2, CSF3R, IL2RA,OSMR, MYD88, IL31RA, IFNAR2, IL6R, CSF2RA, IL13RA2, EPOR, IL1R1, IL10RB,or IL3RA, when present at the first intracellular domain (P3), andconstructs with intracellular domains from CD27, CD40, CD79B, TNFRSF4,TNFRSF18, CD3D, CD3G, CD27, ICOS, TNFRSF9, CD3E, FCGR2A, CD28, CD79A, orFCGR2C, when present at the second intracellular domain (P4), showedparticularly noteworthy enrichments in both Libraries 4B and 4.1B (Table23). Constructs with domains from the cytokine receptors IL18R1, MPL,CRLF2, IL11RA, IL13RA1, IL2RG, IL7R, IFNGR2, CSF3R, IL2RA, OSMR, IL31RA,IFNAR2, IL6R, CSF2RA, IL13RA2, EPOR, IL1R1, IL10RB, or IL3RA, whenpresent at the first intracellular domain (P3), and constructs withdomains from the cytokine receptors CD27, CD40, TNFRSF4, TNFRSF18,TNFRSF9, FCGR2A, or FCGR2C, when present at the second intracellulardomain (P4), showed particularly noteworthy enrichments in bothLibraries 4B and 4.1B (Table 23). Constructs with domains containingITAM motifs from CD79B, CD3D, CD3G, CD3E, FCGR2A, CD79A, or FCGR2C, whenpresent at the second intracellular domain (P4), showed particularlynoteworthy enrichments in both Libraries 4B and 4.1B (Table 23).

In some illustrative embodiments, the CLE comprises an intracellulardomain from a cytokine receptor in any of the constructs of Tables19-23. In some illustrative embodiments, the CLE comprises anintracellular domain from any of the constructs of Tables 19-23 withonly a single intracellular domain (other P3 or P4 was a linker orstop).

Information about various exemplary CLE domains provided herein, isfound in Table 6. For example, the first CLE identified in Table 7 isM008-S212-S075. For this CLE, the extracellular and transmembrane domain(P1-2) is M008, the first intracellular domain (P3) is S212, and thesecond intracellular domain (P4) is S075. Detailed information about theidentity of these domains or modules when considering nucleic acidsencoding the same, for example M008, is found in Table 6. Table 6discloses that M008 is ECDTM-8 and more specifically that this is aninterleukin 7 receptor alpha (IL7RA) mutant with a PPCL insert and thatthe construct includes an eTag.

For Library 1A, intracellular domains from CD3D, CD3E, CD8A, CD27, CD40,CD79B, IFNAR1, IL2RA, IL3RA, IL13RA2, TNFRSF8, and TNFRSF9, or mutantsthereof that are known to have signaling activity, when found at thefirst intracellular domain position (P3) of candidate chimericpolypeptides promoted proliferation of PBMCs between day 7 and the lastday, when data was considered in a combined manner for all constructswith first intracellular domains (P3) derived from that gene.Accordingly, exemplary embodiments of CLEs provided herein haveintracellular domains from CD3D, CD3E, CD8A, CD27, CD40, CD79B, IFNAR1,IL2RA, IL3RA, IL13RA2, TNFRSF8, and TNFRSF9, including mutants thereofthat retain signaling activity, as either the second intracellulardomain, or in illustrative embodiments, the first intracellular domain.

For Library 1A, intracellular domains from CD3D, CD3G, CD8A, CD8B, CD27,CD40, CD79B, CRLF2, FCGR2C, ICOS, IL2RA, IL13RA1, IL13RA2, IL15RA,TNFRSF9, and TNFRSF18, or mutants thereof that are known to havesignaling activity, when found at the second intracellular domainposition (P4) of candidate chimeric polypeptides promoted proliferationof PBMCs between day 7 and the last day, when data was considered in acombined manner for all constructs with a second intracellular domains(P4) derived from that gene. Examples of second intracellular domains orportions and/or mutants thereof, that were present in constructs thatpromoted cell proliferation are provided in tables in Example 17.Accordingly, exemplary embodiments of CLEs provided herein haveintracellular domains from CD3D, CD3G, CD8A, CD8B, CD27, CD40, CD79B,CRLF2, FCGR2C, ICOS, IL2RA, IL13RA1, IL13RA2, IL15RA, TNFRSF9, andTNFRSF18, including mutants thereof that retain signaling activity, aseither the first intracellular domain, or in illustrative embodiments,the second intracellular domain.

For Library 3A, first intracellular (P3) domains from CSF2RA, IFNAR1,ILIRAP, IL4R, IL6ST, IL1IRA, IL12RB2, IL17RA, IL17RD, IL17RE, IL18R1,IL21R, IL23R, MPL, and MyD88, or mutants thereof that are known topromote signaling activity in certain cell types if such mutants arepresent in the constructs provided in Example 18, when found at thefirst intracellular domain (P3) of candidate chimeric polypeptideselements herein, promoted proliferation of PBMCs between day 7 and thelast day or between day 7 and day 21, when data is considered in acombined manner for all constructs with a first intracellular domainderived from that gene. This conclusion is based on enrichment ofsequence counts of constructs in mixed cultured PBMC cell populations,such that enrichment, calculated as the logarithm in base 2 of the ratiobetween normalized count at the last day indicated on the table plus oneand normalized count at day 7 plus one, was at least 2 for Library 3A,when results for all constructs for a gene are combined. For Library 3B,first intracellular domains from CSF2RB, IL4R, and MPL, or mutantsthereof that are known to promote signaling activity in certain celltypes if such mutants are present in the constructs provided in Example18, were the best performers when analyzed in this way. Examples offirst intracellular domains or portions and/or mutants thereof that werepresent in constructs that promoted cell proliferation for Libraries 3A,3B, 3.1A, and 3.1B are provided in Tables 12-15.

For Library 3A, second intracellular (P4) domains from CD3D, CD3G, CD27,CD40, CD79A, CD79B, FCER1G, FCGRA2, ICOS, TNFRSF4, and TNFRSF8, ormutants thereof that are known to promote signaling activity in certaincell types if such mutants are present in the constructs provided inExample 18, when found at the second intracellular domain (P4) ofcandidate chimeric polypeptides elements herein, promoted proliferationof PBMCs between day 7 and day 21 or between day 7 and the last dayindicated on the table, when data is considered in a combined manner forall constructs with a second intracellular domain derived from thatgene. This conclusion is based on enrichment of sequence counts ofconstructs in mixed cultured PBMC cell populations, such thatenrichment, calculated as the logarithm in base 2 of the ratio betweennormalized count at the last day indicated on the table plus one andnormalized count at day 7 plus one, was at least 2 for Library 3A, whenresults for all constructs for a gene are combined. Examples of secondintracellular domains or portions and/or mutants thereof that werepresent in constructs that promoted cell proliferation for Libraries 3Aand 3B are provided in Tables 12 to 17.

After an initial interim decoding analysis, for Library 3A, firstintracellular (P3) domains from IL17RD, IL17RE, ILIRAP, IL23R, and MPL,or mutants thereof that are known to promote signaling activity incertain cell types, when found at the first intracellular domain (P3) ofcandidate chimeric polypeptides elements herein, promoted proliferationof PBMCs between day 7 and day 21, when data is considered in a combinedmanner for all constructs with a first intracellular domain derived fromthat gene. For Library 3B, first intracellular domains from IL21R, MPL,and OSMR, or mutants thereof that are known to promote signalingactivity in certain cell types, were the best performers when analyzedin this way. Examples of first intracellular domains or portions and/ormutants thereof, that were present in constructs that promoted cellproliferation for Libraries 3A and 3B are provided in tables in Example18. Accordingly, exemplary embodiments of CLEs provided herein haveintracellular domains from IL17RD, IL17RE, ILIRAP, IL23R, and MPL F9,including mutants thereof that retain signaling activity, as either thesecond intracellular domain, or in illustrative embodiments, the firstintracellular domain.

After an initial decoding analysis, for Library 3A, second intracellular(P4) domains from CD27, CD3G, CD40, and CE79B, or mutants thereof thatare known to promote signaling activity in certain cell types, whenfound at the second intracellular domain (P4) of candidate chimericpolypeptides elements herein, promoted proliferation of PBMCs betweenday 7 and the last day indicated on the table, when data is consideredin a combined manner for all constructs with a second intracellulardomain derived from that gene. For Library 3B, second intracellulardomains from CD40, or mutants thereof that are known to promotesignaling activity in certain cell types, were the best performance whenanalyzed in this way. Accordingly, exemplary embodiments of CLEsprovided herein have intracellular domains from CD27, CD3G, CD40, andCE79B, including mutants thereof that retain signaling activity, aseither the first intracellular domain, or in illustrative embodiments,the second intracellular domain.

As shown in Tables 12 to 17, first intracellular (P3) domains from MPL,LEPR, MYD88, IFNAR2, or in some cases mutants thereof that retainsignaling activity, when found at the first intracellular domain (P3) ofcandidate chimeric polypeptides herein, promoted proliferation of PBMCsbetween day 7 and the end of the experiment, when considering the firstintracellular domains that occur most frequently in top candidateconstructs. Examples of first intracellular domains or portions and/ormutants thereof, that were present in constructs that promoted cellproliferation for Libraries 3A, 3B, 3.1A, 3, 1B, 4B, and 4.1B areprovided in tables in Example 18. Accordingly, exemplary embodiments ofCLEs provided herein have intracellular domains from MPL, LEPR, MYD88,IFNAR2, including mutants thereof that retain signaling activity, aseither the second intracellular domain, or in illustrative embodiments,the first intracellular domain.

As shown in Tables 12 to 17, second intracellular (P4) domains fromCD40, CD79B, CD27, or in some cases mutants thereof that retainsignaling activity, when found at the second intracellular domain (P4)of candidate chimeric polypeptides herein, promoted proliferation ofPBMCs between day 7 and the last day indicated on the table, whenconsidering the first intracellular domains that occur most frequentlyin top candidate constructs. Examples of first intracellular domains orportions and/or mutants thereof, that were present in constructs thatpromoted cell proliferation for Libraries 3A, 3B, 3.1A, 3, 1B, 4B, and4.1B are provided in tables in Example 18. Accordingly, exemplaryembodiments of CLEs provided herein have intracellular domains fromCD40, CD79B, CD27, including mutants thereof that retain signalingactivity, as either the first intracellular domain, or in illustrativeembodiments, the second intracellular domain.

In noteworthy embodiments of any of the isolated polynucleotide orvector aspects and embodiments provided herein that comprise a firstnucleic acid sequence that encodes a chimeric polypeptide that promotescell proliferation of PBMCs, B cells, T cells, and/or NK cells (i.e. aCLE), and a second nucleic acid sequence that encodes a chimeric antigenreceptor (CAR) comprising an antigen-specific targeting region (ASTR), atransmembrane domain, and an intracellular activating domain, the firstnucleic acid sequence and the second nucleic acid sequence can beseparated by a ribosomal skip sequence. In some embodiments, theribosomal skip sequence is F2A, E2A, P2A, or T2A.

As disclosed herein, lymphoproliferative elements, as exemplified hereinwith CLEs, in some illustrative embodiments can include a dimerizingmotif to help enhance and/or regulate activity thereof, for example toaffect signals in a cell, such as signals that affect protein activityor gene expression. Such dimerizing motif is typically a portion of, orthe entire extracellular domain. In some embodiments, a dimerizingmoiety can be attached to a recognition and clearance sequence in theextracellular domain of a CLE herein. In fact, in some embodiments, alymphoproliferative element can include an entire lymphoproliferativeelement from one protein except that the extracellular domain includes adimerizing moiety.

In some embodiments of lymphoproliferative elements and CLEs thatinclude dimerizing agents, and polynucleotides and nucleic acidsequences encoding the same, the dimerizing motif can include an aminoacid sequence from transmembrane homodimeric polypeptides that naturallyexist as homodimers. For example, the dimerizing motif can be a leucinezipper polypeptide, for example a Jun polypeptide as exemplified inExample 18 herein. In some embodiments, these transmembrane homodimericpolypeptides can include early activation antigen CD69 (CD69),Transferrin receptor protein 1 (CD71), B-cell differentiation antigen(CD72), T-cell surface protein tactile (CD96), Endoglin (Cd105), Killercell lectin-like receptor subfamily B member 1 (Cd161), P-selectinglycoprotein ligand 1 (Cd162), Glutamyl aminopeptidase (Cd249), Tumornecrosis factor receptor superfamily member 16 (CD271), Cadherin-1(E-Cadherin) (Cd324), or active fragments thereof. In some embodiments,the dimerizing motif can include an amino acid sequence fromtransmembrane proteins that dimerize upon ligand (also referred toherein as a dimerizer or dimerizing agent) binding. In some embodiments,the dimerizing motif and dimerizer can include (where the dimerizer isin parentheses following the dimerizer-binding pair): FKBP and FKBP(rapamycin); GyrB and GyrB (coumermycin); DHFR and DHFR (methotrexate);or DmrB and DmrB (AP20187). As noted above, rapamycin can serve as adimerizer. Alternatively, a rapamycin derivative or analog can be used(see, e.g., WO96/41865; WO 99/36553; WO 01/14387; and Ye et al (1999)Science 283:88-91). For example, analogs, homologs, derivatives, andother compounds related structurally to rapamycin (“rapalogs”) include,among others, variants of rapamycin having one or more of the followingmodifications relative to rapamycin: demethylation, elimination orreplacement of the methoxy at C7, C42 and/or C29; elimination,derivatization or replacement of the hydroxy at C13, C43 and/or C28;reduction, elimination or derivatization of the ketone at C14, C24and/or C30; replacement of the 6-membered pipecolate ring with a5-membered prolyl ring; and alternative substitution on the cyclohexylring or replacement of the cyclohexyl ring with a substitutedcyclopentyl ring. Additional information is presented in, e.g., U.S.Pat. Nos. 5,525,610; 5,310,903 5,362,718; and 5,527,907. Selectiveepimerization of the C-28 hydroxyl group has been described (see, e.g.,WO 01/14387). Additional synthetic dimerizing agents suitable for use asan alternative to rapamycin include those described in U.S. PatentPublication No. 2012/0130076. As noted above, coumermycin can serve as adimerizing agent. Alternatively, a coumermycin analog can be used (see,e.g., Farrar et al. (1996) Nature 383:178-181; and U.S. Pat. No.6,916,846). As noted above, in some cases, the dimerizing agent ismethotrexate, e.g., a non-cytotoxic, homo-bifunctional methotrexatedimer (see, e.g., U.S. Pat. No. 8,236,925).

In some embodiments, when present in the plasma membrane of a eukaryoticcell, a lymphoproliferative element, an in illustrative embodiments aCLE, including a dimerizing motif can be active in the absence of adimerizing agent. For example, lymphoproliferative elements that includea leucine zipper-containing dimerization motif, or that include adimerizing motif from transmembrane homodimeric polypeptides includingCD69, CD71, CD72, CD96, Cd105, Cd161, Cd162, Cd249, CD271, Cd324, activemutants thereof, and/or active fragments thereof can be active in theabsence a dimerizing agent. In some embodiments, when present in theplasma membrane of a eukaryotic cell, a lymphoproliferative elementincluding a dimerizing motif can be active in the presence of adimerizing agent. For example, lymphoproliferative elements including adimerizing motif from FKBP, GyrB, DHFR, or DmrB can be active in thepresence of the respective dimerizing agents or analogs thereof, e.g.rapamycin, coumermycin, methotrexate, and AP20187, respectively.

In method embodiments including lymphoproliferative elements, e.g. CLE,that include a dimerizer for dimerizing a polypeptide comprising adimerizing motif,a dimerizer can be administered to the host (subject)using any convenient means capable of resulting in the desired effect.Thus, the dimerizer can be incorporated into a variety of formulationsfor administration. More particularly, a dimerizer can be formulatedinto pharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers or diluents, and may be formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants and aerosols.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific dimerizer, the severity of the symptoms, andthe susceptibility of the subject to side effects. Preferred dosages fora given compound are readily determinable by those of skill in the artby a variety of means.

A dimerizer is administered to an individual using any available methodand route suitable for drug delivery, including in vivo and ex vivomethods, as well as systemic and localized routes of administration.

In any aspects or embodiments wherein the extracellular domain of a CLEcomprises a dimerizing motif, the dimerizing motif can be selected fromthe group consisting of: a leucine zipper motif-containing polypeptide,CD69, CD71, CD72, CD96, Cd105, Cd161, Cd162, Cd249, CD271, and Cd324, aswell as mutants and/or active fragments thereof that retain the abilityto dimerize. In any of the aspects and embodiments herein wherein theextracellular domain of a CLE comprises a dimerizing motif, thedimerizing motif can require a dimerizing agent, and the dimerizingmotif and associated dimerizing agent can be selected from the groupconsisting of: FKBP and rapamycin or analogs thereof, GyrB andcoumermycin or analogs thereof, DHFR and methotrexate or analogsthereof, or DmrB and AP20187 or analogs thereof, as well as mutantsand/or active fragments of the recited dimerizing proteins that retainthe ability to dimerize.

In illustrative embodiments of any aspects or embodiments herein whereinthe extracellular domain of a CLE comprises a dimerizing motif, theextracellular domain can comprises a leucine zipper motif. In someembodiments, the leucine zipper motif is from a jun polypeptide, forexample c-jun. In certain embodiments the c-jun polypeptide is the c-junpolypeptide region of ECD-11.

In any of the aspects and embodiments provided herein that includelymphoproliferative element, the lymphoproliferative element can be apolypeptide comprising a membrane targeting region, a dimerizing (ormultimerizing) domain, a first intracellular domain, and optionally asecond intracellular domain, and further optionally a thirdintracellular domain, and optionally a fourth intracellular domain,wherein at least one of the first and optional second, third, and fourthintracellular domains are from any gene with an intracellular domainidentified in Tables 7 to 17, or selected from any of the first orsecond intracellular domains identified in Tables 7 to 17, and whereinsaid internally dimerizing and/or multimerizing lymphoproliferativeelement promotes cell proliferation of PBMCs, and in illustrativeembodiments, of B cells, T cells, and/or NK cells. Such polypeptide canbe referred to herein as an internally dimerizing and/or multimerizinglymphoproliferative element. In certain embodiments, linkers can beadded between any of the domains. In certain embodiments, the first andsecond intracellular domains are other than MyD88 and CD40. In someembodiments, the intracellular domain is MPL, or an intracellularfragment thereof that retains the ability to promote PBMC cellproliferation. In some embodiments, the intracellular domain is otherthan MPL or an intracellular fragment thereof that retains the abilityto promote PBMC cell proliferation. In some embodiments, theintracellular domain is other than MyD88 or an intracellular fragmentthereof that retains the ability to promote PBMC cell proliferation.

The dimerizing domain of such internally dimerizing and/or multimerizingpolypeptide lymphoproliferative elements in illustrative embodiments islocated between the membrane targeting region and the firstintracellular domain. In certain embodiments the dimerizing (ormultimerizing) domain comprises a multimerizing or dimerizing ligandbinding sites, such as, for example, an FKBP region, for example FKBP12.In some embodiments, the polypeptide can comprise an extracellulardomain, such as any of the extracellular domains provided herein, forexample in Tables 7 to 11 of Example 17 or in Tables 12 to 17 of Example18, with or without the recognition and clearance domain. In certainembodiments, the membrane targeting region is selected from the groupconsisting of a myristoylation region, palmitoylation region,prenylation region, and transmembrane sequences of receptors. In certainembodiments, the membrane targeting region is a myristoylation region.The internally dimerizing and/or multimerizing lymphoproliferativeelement is typically tethered to the plasma membrane via the membranetargeting region, for example tethered to the membrane with anN-terminal myristate. In illustrative embodiments, the FKBP region bindsFK506.

Internally dimerizing and/or multimerizing lymphoproliferative elementsin one embodiment are an integral part of a system that uses a dimericanalog of the lipid permeable immunosuppressant drug, FK506, which losesits normal bioactivity while gaining the ability to crosslink moleculesgenetically fused to the FK506-binding protein, FKBP12. By fusing one ormore FKBPs and a myristoylation sequence to the cytoplasmic signalingdomain of a target receptor, one can stimulate signaling in a dimerizerdrug-dependent, but ligand and ectodomain-independent manner. Thisprovides the system with temporal control, reversibility using monomericdrug analogs, and enhanced specificity. The high affinity ofthird-generation AP20187/AP1903 dimerizer drugs for their bindingdomain, FKBP12 permits specific activation of the recombinant receptorin vivo without the induction of non-specific side effects throughendogenous FKBP12. FKBP12 variants having amino acid substitutions anddeletions, such as FKBP12V36, that bind to a dimerizer drug, may also beused. In addition, the synthetic ligands are resistant to proteasedegradation, making them more efficient at activating receptors in vivothan most delivered protein agents.

Pseudotyping Elements

Many of the methods and compositions provided herein includepseudotyping elements. The pseudotyping of replication incompetentrecombinant retroviral particles with heterologous envelopeglycoproteins typically alters the tropism of a virus and facilitatesthe transduction of host cells. A pseudotyping element as used hereincan include a “binding polypeptide” that includes one or morepolypeptides, typically glycoproteins, that identify and bind the targethost cell, and one or more “fusogenic polypeptides” that mediate fusionof the retroviral and target host cell membranes, thereby allowing aretroviral genome to enter the target host cell. In some embodimentsprovided herein, pseudotyping elements are provided aspolypeptide(s)/protein(s), or as nucleic acid sequences encoding thepolypeptide(s)/protein(s).

In some embodiments, the pseudotyping element is the feline endogenousvirus (RD114) envelope protein, the oncoretroviral amphotropic envelopeprotein, the oncoretroviral ecotropic envelope protein, the vesicularstomatitis virus envelope protein (VSV-G), and/or the paramyxovirusMeasles envelope proteins H and F.

In some embodiments, the pseudotyping elements include a bindingpolypeptide and a fusogenic polypeptide derived from different proteins.For example, the replication incompetent recombinant retroviralparticles of the methods and compositions disclosed herein can bepseudotyped with the fusion (F) and hemagglutinin (H) polypeptides ofthe measles virus (MV), as non-limiting examples, clinical wildtypestrains of MV, and vaccine strains including the Edmonston strain(MV-Edm) or fragments thereof. Not to be limited by theory, bothhemagglutinin (H) and fusion (F) polypeptides are believed to play arole in entry into host cells wherein the H protein binds MV toreceptors CD46, SLAM, and Nectin-4 on target cells and F mediates fusionof the retroviral and host cell membranes. In an illustrativeembodiment, especially where the target cell is a T cell and/or NK cell,the binding polypeptide is a Measles Virus H polypeptide and thefusogenic polypeptide is a Measles Virus F polypeptide.

In some studies, lentiviral particles pseudotyped with truncated F and Hpolypeptides had a significant increase in titers and transductionefficiency (Funke et al. 2008. Molecular Therapy. 16(8):1427-1436),(Frecha et al. 2008. Blood. 112(13):4843-4852). The highest titers wereobtained when the F cytoplasmic tail was truncated by 30 residues(referred to as MV(Ed)-FA30 (SEQ ID NO:105)). For the H variants,optimal truncation occurred when 18 or 19 residues were deleted(MV(Ed)-HA18 (SEQ ID NO:106) or MV(Ed)-HA19), although variants with atruncation of 24 residues with and without replacement of deletedresidues with alanine (MV(Ed)-HA24 (SEQ ID NO:235) and MV(Ed)-HA24+A)also resulted in optimal titers.

In some embodiments, including those directed to transducing T cellsand/or NK cells, the replication incompetent recombinant retroviralparticles of the methods and compositions disclosed herein arepseudotyped with mutated or variant versions of the measles virus fusion(F) and hemagglutinin (H) polypeptides, in illustrative examples,cytoplasmic domain deletion variants of measles virus F and Hpolypeptides. In some embodiments, the mutated F and H polypeptides are“truncated H” or “truncated F” polypeptides, whose cytoplasmic portionhas been truncated, i.e. amino acid residues (or coding nucleic acids ofthe corresponding nucleic acid molecule encoding the protein) have beendeleted. “HAY” and “FΔX” designate such truncated H and F polypeptide,respectively, wherein “Y” refers to 1-34 residues that have been deletedfrom the amino termini and “X” refers to 1-35 residues that have beendeleted from the carboxy termini of the cytoplasmic domains. In afurther embodiment, the “truncated F polypeptide” is FΔ24 or FΔ30 and/orthe “truncated H protein” is selected from the group consisting of HA14,HA15, HΔ16, HΔ17, HΔ18, HΔ19, HΔ20, HΔ21+A, HΔ24 and HΔ24+4A, morepreferably HΔ18 or HΔ24. In an illustrative embodiment, the truncated Fpolypeptide is MV(Ed)-FΔ30 and the truncated H polypeptide isMV(Ed)-HΔ18.

In some embodiments, the fusogenic polypeptide includes multipleelements expressed as one polypeptide. In some embodiments, the bindingpolypeptide and fusogenic polypeptide are translated from the sametranscript but from separate ribosome binding sites; in otherembodiments, the binding polypeptide and fusogenic polypeptide areseparated by a cleavage peptide site, which not to be bound by theory,is cleaved after translation, as is common in the literature, or aribosomal skip sequence. In some embodiments, the translation of thebinding polypeptide and fusogenic polypeptide from separate ribosomebinding sites results in a higher amount of the fusogenic polypeptide ascompared to the binding polypeptide. In some embodiments, the ratio ofthe fusogenic polypeptide to the binding polypeptide is at least 2:1, atleast 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, or atleast 8:1. In some embodiments, the ratio of the fusogenic polypeptideto the binding polypeptide is between 1.5:1, 2:1, or 3:1, on the low endof the range, and 3:1, 4:1, 5:1, 6:1, 7:1, 8:1. 9:1 or 10:1 on the highend of the range.

Activation Elements

Many of the methods and composition aspects of the present disclosureinclude an activation element, also referred to herein as a T cellactivation element, or a nucleic acid encoding an activation element.The restrictions associated with lentiviral (LV) transduction intoresting T cells are attributed to a series of pre-entry and post-entrybarriers as well as cellular restrictive factors (Strebel et al 2009.BMC Medicine 7:48). One restriction is the inability for the envelopepseudotyped-LV particles to recognize potential receptors and mediatefusion with the cellular membrane. However, under certain conditions,the transduction of resting T cells with HIV-1-based lentiviral vectorsis possible mostly upon T cell receptor (TCR) CD3 complex and CD28co-stimulation (Korin & Zack. 1998. Journal of Virology. 72:3161-8,Maurice et al. 2002. Blood 99:2342-50), as well as through exposure tocytokines (Cavalieri et al 2003).

Cells of the immune system such as T lymphocytes recognize and interactwith specific antigens through receptors or receptor complexes which,upon recognition or an interaction with such antigens, cause activationof the cell and expansion in the body. An example of such a receptor isthe antigen-specific T lymphocyte receptor complex (TCR/CD3). The T cellreceptor (TCR) is expressed on the surface of T lymphocytes. Onecomponent, CD3, is responsible for intracellular signaling followingoccupancy of the TCR by ligand. The T lymphocyte receptor forantigen-CD3 complex (TCR/CD3) recognizes antigenic peptides that arepresented to it by the proteins of the major histocompatibility complex(MHC). Complexes of MHC and peptide are expressed on the surface ofantigen presenting cells and other T lymphocyte targets. Stimulation ofthe TCR/CD3 complex results in activation of the T lymphocyte and aconsequent antigen-specific immune response. The TCR/CD3 complex plays acentral role in the effector function and regulation of the immunesystem. Thus, activation elements provided herein, activate T cells bybinding to one or more components of the T cell receptor associatedcomplex, for example by binding to CD3. In some cases, the activationelement can activate alone. In other cases, the activation requiresactivation through the TCR receptor complex in order to further activatecells.

T lymphocytes also require a second, co-stimulatory signal to becomefully active. Without such a signal, T lymphocytes are eithernon-responsive to antigen binding to the TCR, or become anergic. Such aco-stimulatory signal, for example, is provided by CD28, a T lymphocyteprotein, which interacts with CD80 and CD86 on antigen-producing cells.As used herein, a functional extracellular fragment of CD80 retains itsability to interact with CD28. ICOS (Inducible COStimulator), another Tlymphocyte protein, provides a co-stimulatory signal when bound to ICOSligand.

Activation of the T cell receptor (TCR) CD3 complex and co-stimulationwith CD28 can occur by ex vivo exposure to solid surfaces (e.g. beads)coated with anti-CD3 and anti-CD28. In some embodiments of the methodsand compositions disclosed herein, resting T cells are activated byexposure to solid surfaces coated with anti-CD3 and anti-CD28 ex vivo.In other embodiments, resting T cells or NK cells, illustrativeembodiments T cells, are activated by exposure to soluble anti-CD3antibodies (e.g. at 50-150, or 75-125, or 100 ng/ml). In suchembodiments, which can be part of methods for genetically modifying ortransducing, in illustrative embodiments without prior activation, suchactivation and/or contacting can be carried out, for example, for 8hours or less, 4 hours or less or between 2 and 8 hours, 2 and 4 hours,or between 2 and 3.

In certain illustrative embodiments of the methods and compositionsprovided herein, polypeptides that are capable of binding CD3 and/orCD28, are presented as “activation elements” on the surface ofreplication incompetent recombinant retroviral particles of the methodsand compositions disclosed herein, which are also aspects of theinvention. Polypeptides that bind CD3 and/or CD28 are referred to as“activation elements” because of their ability to activate resting Tcells.

In some embodiments, the activation element is a polypeptide capable ofbinding to CD3. In some embodiments, the polypeptide capable of bindingto CD3 is an anti-CD3 antibody, or a fragment thereof that retains theability to bind to CD3. In illustrative embodiments, the anti-CD3antibody or fragment thereof is a single chain anti-CD3 antibody, suchas but not limited to, an anti-CD3 scFv. In another illustrativeembodiment, the polypeptide capable of binding to CD3 is anti-CD3scFvFc.

A number of anti-human CD3 monoclonal antibodies and antibody fragmentsthereof are available, and can be used in the present invention,including but not limited to UCHT1, OKT-3, HIT3A, TRX4, X35-3, VIT3,BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111409, CLB-T3.4.2, TR-66,WT31, WT32, SPv-T3b, I1D8, XIII-141, X11146, XIII-87, 12F6, T3/RW2-8C8,T3/RW24B6, OKT3D, M-T301, SMC2 and F101.01.

In some embodiments, the activation element is a polypeptide capable ofbinding to CD28. In some embodiments, the polypeptide capable of bindingto CD28 is an anti-CD28 antibody, or a fragment thereof that retains theability to bind to CD28. In other embodiments, the polypeptide capableof binding to CD28 is CD80, CD86, or a functional fragment thereof thatis capable of binding CD28 and inducing CD28-mediated activation of Akt,such as an external fragment of CD80. In some aspects herein, anexternal fragment of CD80 means a fragment that is typically present onthe outside of a cell in the normal cellular location of CD80, thatretains the ability to bind to CD28. In illustrative embodiments, theanti-CD28 antibody or fragment thereof is a single chain anti-CD28antibody, such as, but not limited to, an anti-CD28 scFv. In anotherillustrative embodiment, the polypeptide capable of binding to CD28 isCD80, or a fragment of CD80 such as an external fragment of CD80.

Anti-CD28 antibodies are known in the art and can include, asnon-limiting examples, monoclonal antibody 9.3, an IgG2a antibody (Dr.Jeffery Ledbetter, Bristol Myers Squibb Corporation, Seattle, Wash.),monoclonal antibody KOLT-2, an IgG1 antibody, 15E8, an IgG1 antibody,248.23.2, an IgM antibody and EX5.3D10, an IgG2a antibody.

In an illustrative embodiment, an activation element includes twopolypeptides, a polypeptide capable of binding to CD3 and a polypeptidecapable of binding to CD28.

In certain embodiments, the polypeptide capable of binding to CD3 orCD28 is an antibody, a single chain monoclonal antibody or an antibodyfragment, for example a single chain antibody fragment. Accordingly, theantibody fragment can be, for example, a single chain fragment variableregion (scFv), an antibody binding (Fab) fragment of an antibody, asingle chain antigen-binding fragment (scFab), a single chainantigen-binding fragment without cysteines (scFabAC), a fragmentvariable region (Fv), a construct specific to adjacent epitopes of anantigen (CRAb), or a single domain antibody (VH or VL).

In any of the embodiments disclosed herein, an activation element, or anucleic acid encoding the same, can include a dimerizing or higher ordermultimerizing motif. Dimerizing and multimerizing motifs are well-knownin the art and a skilled artisan will understand how to incorporate theminto the polypeptides for effective dimerization or multimerization. Forexample, in some embodiments, the activation element that includes adimerizing motif can be one or more polypeptides capable of binding toCD3 and/or CD28. In some embodiments, the polypeptide capable of bindingto CD3 is an anti-CD3 antibody, or a fragment thereof that retains theability to bind to CD3. In illustrative embodiments, the anti-CD3antibody or fragment thereof is a single chain anti-CD3 antibody, suchas but not limited to, an anti-CD3 scFv. In another illustrativeembodiment, the polypeptide capable of binding to CD3 is anti-CD3scFvFc,which in some embodiments is considered an anti-CD3 with a dimerizingmotif without any additional dimerizing motif, since anti-CD3scFvFcconstructs are known to be capable of dimerizing without the need for aseparate dimerizing motif.

In some embodiments, the dimerizing or multimerizing motif, or a nucleicacid sequence encoding the same, can be an amino acid sequence fromtransmembrane polypeptides that naturally exist as homodimers ormultimers. In some embodiments, the dimerizing or multimerizing motif,or a nucleic acid sequence encoding the same, can be an amino acidsequence from a fragment of a natural protein or an engineered protein.In one embodiment, the homodimeric polypeptide is a leucine zippermotif-containing polypeptide (leucine zipper polypeptide). For example,a leucine zipper polypeptide derived from c-JUN, non-limiting examplesof which are disclosed related to chimeric lymphoproliferative elements(CLEs) herein.

In some embodiments, these transmembrane homodimeric polypeptides caninclude early activation antigen CD69 (CD69), Transferrin receptorprotein 1 (CD71), B-cell differentiation antigen (CD72), T-cell surfaceprotein tactile (CD96), Endoglin (Cd105), Killer cell lectin-likereceptor subfamily B member 1 (Cd161), P-selectin glycoprotein ligand 1(Cd162), Glutamyl aminopeptidase (Cd249), Tumor necrosis factor receptorsuperfamily member 16 (CD271), Cadherin-1 (E-Cadherin) (Cd324), oractive fragments thereof. In some embodiments, the dimerizing motif, andnucleic acid encoding the same, can include an amino acid sequence fromtransmembrane proteins that dimerize upon ligand (also referred toherein as a dimerizer or dimerizing agent) binding. In some embodiments,the dimerizing motif and dimerizer can include (where the dimerizer isin parentheses following the dimerizer-binding pair): FKBP and FKBP(rapamycin); GyrB and GyrB (coumermycin); DHFR and DHFR (methotrexate);or DmrB and DmrB (AP20187). As noted above, rapamycin can serve as adimerizer. Alternatively, a rapamycin derivative or analog can be used(see, e.g., WO96/41865; WO 99/36553; WO 01/14387; and Ye et al (1999)Science 283:88-91). For example, analogs, homologs, derivatives, andother compounds related structurally to rapamycin (“rapalogs”) include,among others, variants of rapamycin having one or more of the followingmodifications relative to rapamycin: demethylation, elimination orreplacement of the methoxy at C7, C42 and/or C29; elimination,derivatization or replacement of the hydroxy at C13, C43 and/or C28;reduction, elimination or derivatization of the ketone at C14, C24and/or C30; replacement of the 6-membered pipecolate ring with a5-membered prolyl ring; and alternative substitution on the cyclohexylring or replacement of the cyclohexyl ring with a substitutedcyclopentyl ring. Additional information is presented in, e.g., U.S.Pat. Nos. 5,525,610; 5,310,903 5,362,718; and 5,527,907. Selectiveepimerization of the C-28 hydroxyl group has been described (see, e.g.,WO 01/14387). Additional synthetic dimerizing agents suitable for use asan alternative to rapamycin include those described in U.S. PatentPublication No. 2012/0130076. As noted above, coumermycin can serve as adimerizing agent. Alternatively, a coumermycin analog can be used (see,e.g., Farrar et al. (1996) Nature 383:178-181; and U.S. Pat. No.6,916,846). As noted above, in some cases, the dimerizing agent ismethotrexate, e.g., a non-cytotoxic, homo-bifunctional methotrexatedimer (see, e.g., U.S. Pat. No. 8,236,925).

In some embodiments, when present on the surface of replicationincompetent recombinant retroviral particles, an activation elementincluding a dimerizing motif can be active in the absence of adimerizing agent. For example, activation elements including adimerizing motif from transmembrane homodimeric polypeptides includingCD69, CD71, CD72, CD96, Cd105, Cd161, Cd162, Cd249, CD271, Cd324, activemutants thereof, and/or active fragments thereof can be active in theabsence a dimerizing agent. In some embodiments, the activation elementcan be an anti-CD3 single chain fragment and include a dimerizing motifselected from the group consisting of CD69, CD71, CD72, CD96, Cd105,Cd161, Cd162, Cd249, CD271, Cd324, active mutants thereof, and/or activefragments thereof.

In some embodiments, when present on the surface of replicationincompetent recombinant retroviral particles, an activation elementincluding a dimerizing motif can be active in the presence of adimerizing agent. For example, activation elements including adimerizing motif from FKBP, GyrB, DHFR, or DmrB can be active in thepresence of the respective dimerizing agents or analogs thereof, e.g.rapamycin, coumermycin, methotrexate, and AP20187, respectively. In someembodiments, the activation element can be a single chain antibodyfragment against anti-CD3 or anti-CD28, or another molecule that bindsCD3 or CD28, and the dimerizing motif and dimerizing agent can beselected from the group consisting of FKBP and rapamycin or analogsthereof, GyrB and coumermycin or analogs thereof, DHFR and methotrexateor analogs thereof, or DmrB and AP20187 or analogs thereof.

In some embodiments, an activation element is fused to a heterologoussignal sequence and/or a heterologous membrane attachment sequence, bothof which help direct the activation element to the membrane. Theheterologous signal sequence targets the activation element to theendoplasmic reticulum, where the heterologous membrane attachmentsequence covalently attaches to one or several fatty acids (also knownas posttranslational lipid modification) such that the activationelements that are fused to the heterologous membrane attachment sequenceare anchored in the lipid rafts of the plasma membrane. In someembodiments, posttranslational lipid modification can occur viamyristoylation, palmitoylation, or GPI anchorage. Myristoylation is apost-translational protein modification which corresponds to thecovalent linkage of a 14-carbon saturated fatty acid, the myristic acid,to the N-terminal glycine of a eukaryotic or viral protein.Palmitoylation is a post-translational protein modification whichcorresponds to the covalent linkage of a C16 acyl chain to cysteines,and less frequently to serine and threonine residues, of proteins. GPIanchorage refers to the attachment of glycosylphosphatidylinositol, orGPI, to the C-terminus of a protein during posttranslationalmodification.

In some embodiments, the heterologous membrane attachment sequence is aGPI anchor attachment sequence. The heterologous GPI anchor attachmentsequence can be derived from any known GPI-anchored protein (reviewed inFerguson M A J, Kinoshita T, Hart G W. GlycosylphosphatidylinositolAnchors. In: Varki A, Cummings R D, Esko J D, et al., editors.Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (NY): ColdSpring Harbor Laboratory Press; 2009. Chapter 11). In some embodiments,the heterologous GPI anchor attachment sequence is the GPI anchorattachment sequence from CD14, CD16, CD48, CD55 (DAF), CD59, CD80, andCD87. In some embodiments, the heterologous GPI anchor attachmentsequence is derived from CD16. In illustrative embodiments, theheterologous GPI anchor attachment sequence is derived from Fc receptorFcγRIIIb (CD16b) or decay accelerating factor (DAF), otherwise known ascomplement decay-accelerating factor or CD55.

In some embodiments, one or both of the activation elements include aheterologous signal sequence to help direct expression of the activationelement to the cell membrane. Any signal sequence that is active in thepackaging cell line can be used. In some embodiments, the signalsequence is a DAF signal sequence. In illustrative embodiments, anactivation element is fused to a DAF signal sequence at its N terminusand a GPI anchor attachment sequence at its C terminus.

In an illustrative embodiment, the activation element includes anti-CD3scFvFc fused to a GPI anchor attachment sequence derived from CD14 andCD80 fused to a GPI anchor attachment sequence derived from CD16b; andboth are expressed on the surface of a replication incompetentrecombinant retroviral particle provided herein. In some embodiments,the anti-CD3 scFvFc is fused to a DAF signal sequence at its N terminusand a GPI anchor attachment sequence derived from CD14 at its C terminusand the CD80 is fused to a DAF signal sequence at its N terminus and aGPI anchor attachment sequence derived from CD16b at its C terminus; andboth are expressed on the surface of a replication incompetentrecombinant retroviral particle provided herein. In some embodiments,the DAF signal sequence includes amino acid residues 1-30 of the DAFprotein.

Membrane-Bound Cytokines

Some embodiments of the method and composition aspects provided herein,include a membrane-bound cytokine, or polynucleotides encoding amembrane-bound cytokine. Cytokines are typically, but not always,secreted proteins. Cytokines that are naturally secreted can beengineered as fusion proteins to be membrane-bound. Membrane-boundcytokine fusion polypeptides are included in methods and compositionsdisclosed herein, and are also an aspect of the invention. In someembodiments, replication incompetent recombinant retroviral particleshave a membrane-bound cytokine fusion polypeptide on their surface thatis capable of binding a T cell and/or NK cell and promotingproliferation and/or survival thereof. Typically, membrane-boundpolypeptides are incorporated into the membranes of replicationincompetent recombinant retroviral particles, and when a cell istransduced by the replication incompetent recombinant retroviralparticles, the fusion of the retroviral and host cell membranes resultsin the polypeptide being bound to the membrane of the transduced cell.

In some embodiments, the cytokine fusion polypeptide includes IL-7,IL-15, or an active fragment thereof. The membrane-bound cytokine fusionpolypeptides are typically a cytokine fused to heterologous signalsequence and/or a heterologous membrane attachment sequence. In someembodiments, the heterologous membrane attachment sequence is a GPIanchor attachment sequence. The heterologous GPI anchor attachmentsequence can be derived from any known GPI-anchored protein (reviewed inFerguson MAJ, Kinoshita T, Hart G W. GlycosylphosphatidylinositolAnchors. In: Varki A, Cummings R D, Esko J D, et al., editors.Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (NY): ColdSpring Harbor Laboratory Press; 2009. Chapter 11). In some embodiments,the heterologous GPI anchor attachment sequence is the GPI anchorattachment sequence from CD14, CD16, CD48, CD55 (DAF), CD59, CD80, andCD87. In some embodiments, the heterologous GPI anchor attachmentsequence is derived from CD16. In an illustrative embodiment, theheterologous GPI anchor attachment sequence is derived from Fc receptorFcγRIIIb (CD16b). In some embodiments, the GPI anchor is the GPI anchorof DAF.

In illustrative embodiments, the membrane-bound cytokine is a fusionpolypeptide of a cytokine fused to DAF. DAF is known to accumulate inlipid rafts that are incorporated into the membranes of replicationincompetent recombinant retroviral particles budding from packagingcells. Accordingly, not to be limited by theory, it is believed that DAFfusion proteins are preferentially targeted to portions of membranes ofpackaging cells that will become part of a recombinant retroviralmembrane.

In non-limiting illustrative embodiments, the cytokine fusionpolypeptide is an IL-7, or an active fragment thereof, fused to DAF. Ina specific non-limiting illustrative embodiment, the fusion cytokinepolypeptide includes in order: the DAF signal sequence (residues 1-31 ofDAF), IL-7 without its signal sequence, and residues 36-525 of DAF.

Riboswitch Control Element Riboswitches

Some of the compositions and methods provided herein include one or moreriboswitches or polynucleotides that include one or more riboswitch,which themselves form distinct aspects of the present disclosure.Riboswitches are a common feature in bacteria to regulate geneexpression and are a means to achieve RNA control of biologicalfunctions. Riboswitches are polynucleotides that can be present in the5′-untranslated region of mRNAs and allow for regulatory control overgene expression through binding of a small molecule ligand that inducesor suppresses a riboswitch activity. Typically, the riboswitch controlsa gene product involved in the generation of the small molecule ligand,thus forming a feedback loop. Riboswitches typically act in acis-fashion, although riboswitches have been identified that act in atrans-fashion. Natural riboswitches consist of two domains: an aptamerdomain that binds the ligand through a three-dimensional folded RNAstructure and a function switching domain that induces or suppresses anactivity in the riboswitch based on the absence or presence of theligand. Thus, there are two ligand sensitive conformations achieved bythe riboswitch, representing on and off states (Garst et al., 2011). Thefunction switching domain can affect the expression of a polynucleotideby regulating: an internal ribosome entry site, pre-mRNA splice donoraccessibility in the retroviral gene construct, pre-mRNA splicing,translation, termination of transcription, transcript degradation, miRNAexpression, or shRNA expression (Dambach and Winkler 2009). The aptamerand function switching domains can be used as modular componentsallowing for synthetic RNA devices to control gene expression either asnative aptamers, mutated/evolved native aptamers, or totally syntheticaptamers that are identified from screening random RNA libraries(McKeague et al 2016).

The purine riboswitch family represents one of the largest families withover 500 sequences found (Mandal et al 2003; US20080269258; andWO2006055351). The purine riboswitches share a similar structureconsisting of three conserved helical elements/stem structures (P1, P2,P3) with intervening loop/junction elements (J1-2, L2, J2-3, L3, J3-1).The aptamer domains of the purine family of riboswitches naturally varyin their affinity/regulation by various purine compounds such asadenine, guanine, adenosine, guanosine, deoxyadenosine, deoxyguanosine(FIG. 5 ), etc. due to sequence variation (Kim et al. 2007).

In one aspect, provided herein is an isolated polynucleotide forregulating expression of a target polynucleotide, including: apolynucleotide encoding the target polynucleotide operably linked to apromoter and a riboswitch, wherein the riboswitch includes: a.) anaptamer domain capable of binding a nucleoside analogue antiviral drugand having reduced binding to guanine or 2′-deoxyguanosine relative tothe nucleoside analogue antiviral drug; and b.) a function switchingdomain capable of regulating expression of the target polynucleotide,wherein binding of the nucleoside analogue by the aptamer domain inducesor suppresses the expression regulating activity of the functionswitching domain, thereby regulating expression of the target gene. Insome embodiments, the target polynucleotide can be a polypeptideencoding region, an miRNA, or an shRNA. In a non-limiting example, theriboswitch is operably linked to a nucleic acid encoding a polypeptide,miRNA, or shRNA with in vivo activity, for example that is effective attreating a disease. For example, in such a non-limiting example, theriboswitch is operably linked to a nucleic acid encoding a chimericantigen receptor. In non-limiting illustrative examples provided herein,the target polynucleotide encodes one or more engineered signalingpolypeptides included in various other aspects of the presentdisclosure. In these non-limiting illustrative examples, the riboswitchand the target polynucleotide encoding one or more engineered signalingpolypeptides can be found in the genome of a packaging cell, in areplication incompetent recombinant retroviral particle, in a T celland/or in an NK cell.

In some embodiments, the aptamer domain can be between 30, 35, 40, 45,50, 55, 60, 65, and 70 nucleotides in length on the low end of the rangeand 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100nucleotides in length on the high end of the range, for example between45 and 80 nucleotides in length, between 45 and 60 nucleotides inlength, or between 45 and 58 nucleotides in length. In illustrativeembodiments, the nucleoside analogue antiviral drug can be thepharmaceutical ligand acyclovir (also known as aciclovir andacycloguanosine) or penciclovir (FIG. 5 ). In some embodiments, theaptamer domain can have a binding affinity to the nucleoside analogueantiviral drug greater than, for example at least 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100-fold greater than thebinding affinity to the nucleoside or nucleotide.

The control element, for example a riboswitch, in some embodiments isoperably linked to a target gene and can control expression of thetarget gene in vitro and/or in vivo. promotes expansion of transduced Tcells in vivo. In some embodiments, expansion is dependent on thepresence of the control element. However, in other embodiments,expansion of the transduced T cells can be at least partially driven byother factors such as the presence of interleukins within the subjectand binding of the ASTR of a CAR on the recombinant T cell to itsligand.

In some embodiments, a nucleoside analogue antiviral drug, for exampleacyclovir or penciclovir, is administered to a subject before, during,and/or after PBLs are isolated from the blood and before T cells and/orNK cells are contacted with a replication incompetent recombinantretroviral particle that includes a control element, which inillustrative non-limiting examples is a riboswitch, that binds to thenucleoside analogue antiviral drug and regulates expression of one ormore target polynucleotides. The one or more target polynucleotides canencode one or more polypeptides that in non-limiting illustrativeexamples are one or more engineered signaling polypeptides, at least oneof which encodes at least one lymphoproliferative element. In someembodiments, the nucleoside analogue antiviral drug, for exampleacyclovir or penciclovir, is administered to the subject for between 5,10, 15, 30, and 60 minutes on the low end of the range, and 1.5, 2, 3,4, 5, 6, 8, 12, 24, 48, or 72 hours on the high end of the range, beforePBLs are isolated from the blood or before T cells and/or NK cells arecontacted with replication incompetent recombinant retroviral particles.In some embodiments, the nucleoside analogue antiviral drug, for exampleacyclovir or penciclovir, is administered to the subject for between1.5, 2, 3, 4, 5, 6, 8, 12, or 24 hours on the low end of the range, ½,1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 days on the high end of therange, after PBLs are isolated from the blood or after T cells and/or NKcells are contacted with replication incompetent recombinant retroviralparticles in methods provided herein. In some embodiments, thenucleoside analogue antiviral drug, for example acyclovir orpenciclovir, is administered to the subject for at least 1.5, 2, 3, 4,5, 6, 8, 12, or 24 hours, or at least 2, 3, 4, 5, 6, 7, 10, 14, 21, or28 days after PBLs are isolated from the blood or after T cells and/orNK cells are contacted with replication incompetent recombinantretroviral particles in methods provided herein. In some embodiments,the nucleoside analogue antiviral drug, for example acyclovir orpenciclovir, is administered to the subject for at least 1, 2, 3, 4, 5,7, 10, 14, 21, 28, 30, 60, 90, or 120 days or 5, 6, 9, 12, 24, 36, 48,60, 72, 84, 96, 120 months or indefinitely after the PBLs have beenreinfused into the subject. In any of the embodiments disclosed herein,the nucleoside analogue antiviral drug can be administered before and/orduring the reinfusion of the PBLs and/or after the PBLs have beenreinfused. In some embodiments, the nucleoside analogue antiviral drugis administered until a subject no longer experiences symptoms of, or isafflicted by, a disease for which the target polynucleotide is related.

In some embodiments, the aptamer domain can preferentially bindpenciclovir over acyclovir or alternatively another antiviral agent,such that concomitant antiviral therapy may be utilized withoutaffecting the riboswitch. In some embodiments, the aptamer domain canbind penciclovir with a binding affinity greater than, for example atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or100-fold greater than the aptamer domain binds acyclovir or anotherantiviral agent. In some embodiments, the aptamer domain canpreferentially bind acyclovir over penciclovir or alternatively anotherantiviral agent, such that concomitant antiviral therapy may be utilizedwithout affecting the riboswitch. In some embodiments, the aptamerdomain can bind acyclovir with a binding affinity greater than, forexample at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80,90, or 100-fold greater than the aptamer domain binds penciclovir oranother antiviral agent. In some embodiments, the oral prodrugs ofpenciclovir (famciclovir) and acyclovir (valaciclovir) can be given to asubject.

In some embodiments, the aptamer domain of an isolated polynucleotidecan share at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity or be identical to any one of thesequences of SEQ ID NOs:87-93 and retain the ability to bind acyclovirand a reduced ability to bind to guanine or 2′-deoxyguanosine relativeto the nucleoside analogue antiviral drug, and wherein the aptamerdomain retains the ability to induce or suppress the expressionregulating activity of the function switching domain when bound byacyclovir. In some embodiments, the aptamer domain of an isolatedpolynucleotide can share at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity or be identical to theaptamer domain of SEQ ID NOs:94-100 and retain the ability to bindpenciclovir and a reduced ability to bind to guanine or2′-deoxyguanosine relative to the nucleoside analogue antiviral drug,and wherein the aptamer domain retains the ability to induce or suppressthe expression regulating activity of the function switching domain whenbound by penciclovir. In some embodiments, a region of an isolatedpolynucleotide or a region of a riboswitch can share at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity orbe identical to any one of the sequences of SEQ ID NOs:87-100.

In some embodiments, a DNA sequence containing a region of an aptamerdomain of an isolated polynucleotide can share at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or beidentical to any one of the sequences of SEQ ID NOs:108-221. In someembodiments, a region of an isolated polynucleotide can share at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity or be identical to any one of the sequences of SEQ IDNOs:108-221.

In some embodiments, a DNA sequence containing a region of an aptamerdomain of an isolated polynucleotide can share at least 80%, 85%, 90%,91%, 91.84%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identityor be identical to SEQ ID NO:108. In some embodiments, a DNA sequencecontaining a region of an aptamer domain of an isolated polynucleotidecan share at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95.83%, 96%,97%, 98%, or 99% sequence identity or be identical to SEQ ID NO:147. Insome embodiments, a DNA sequence containing a region of an aptamerdomain of an isolated polynucleotide can share at least 80%, 85%, 90%,91%, 92%, 93%, 93.88%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identityor be identical to SEQ ID NO:164. In some embodiments, a DNA sequencecontaining a region of an aptamer domain of an isolated polynucleotidecan share at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95.83%96%,97%, 98%, or 99% sequence identity or be identical to SEQ ID NO:183. Insome embodiments, a DNA sequence containing a region of an aptamerdomain of an isolated polynucleotide can share at least 80%, 85%, 90%,91%, 91.84%, 92%, 93%, 94%, 95%, 95.83%96%, 97%, 98%, or 99% sequenceidentity or be identical to SEQ ID NO:198.

In some embodiments, a region of an isolated polynucleotide can includeany one of the consensus sequences of SEQ ID NOs:222-226. In someembodiments, a region of an isolated polynucleotide can share at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95.83%, 96%, 97%, 98%, or 99%sequence identity or be identical to any one of the sequences of SEQ IDNOs:222-226.

In any of the embodiments disclosed herein, the isolated polynucleotidecan retain the ability to bind acyclovir and/or penciclovir. In any ofthe embodiments disclosed herein, an isolated polynucleotide can be thereverse complement of any one of the sequences of SEQ ID NOs: 87-100 orSEQ ID NOs:108-221. In any of the embodiments disclosed herein, anisolated polynucleotide can be a transcription or RNA version of eitherthe DNA sequences of SEQ ID NOs:108-221 or the DNA sequencescomplementary to SEQ ID NOs:108-221. In any of the embodiments disclosedherein, an isolated polynucleotide can be a reverse transcription or DNAversion of any one of the RNA sequences of SEQ ID NOs:87-100 or the DNAstrand complementary to a reverse transcription of any one of the RNAsequences of SEQ ID NOs:87-100.

In some embodiments provided herein, riboswitch scaffolds can be usedfor mutational analysis or molecular evolution. The riboswitchesselected for mutational analysis or molecular evolution can be from anyknown organism, for example, bacteria. In some embodiments, the type I-Adeoxyguanosine riboswitch from Mesoplasma florum can be used formolecular evolution. In some embodiments, the derived aptamer domain canbe at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% identical to theaptamer domain from the type I-A deoxyguanosine riboswitch fromMesoplasma florum (SEQ ID NO:237). In other embodiments, the xptriboswitch from Bacillus subtilis can be used. In some embodiments, thederived aptamer domain can be at least 50%, 60%, 70%, 75%, 80%, 85%,90%, or 95% identical to the aptamer domain from the xpt riboswitch fromBacillus subtilis (SEQ ID NO:243).

The aptamer domains can be used as modular components and combined withany of the function switching domains to affect the RNA transcript. Inany of the embodiments disclosed herein, the riboswitch can affect theRNA transcript by regulating any of the following activities: internalribosomal entry site (IRES), pre-mRNA splice donor accessibility,pre-mRNA splicing, translation, termination of transcription, transcriptdegradation, miRNA expression, or shRNA expression. In some embodiments,the function switching domain can control binding of an anti-IRES to anIRES (see, e.g. Ogawa, RNA (2011), 17:478-488, the disclosure of whichis incorporated by reference herein in its entirety). In any of theembodiments disclosed herein, the presence or absence of the smallmolecule ligand can cause the riboswitch to affect the RNA transcript.In some embodiments, the riboswitch can include a ribozyme. Riboswitcheswith ribozymes can inhibit or enhance transcript degradation of targetpolynucleotides in the presence of the small molecule ligand. In someembodiments, the ribozyme can be a pistol class of ribozyme, ahammerhead class of ribozyme, a twisted class of ribozyme, a hatchetclass of ribozyme, or the HDV (hepatitis delta virus) ribozyme.

In any of the embodiments disclosed herein, the riboswitch can belocated in various positions relative to the target polynucleotide, asis known generally for riboswitches. In some embodiments, the riboswitchcan regulate pre-mRNA splice donor accessibility or pre-mRNA splicingand be located before the target polynucleotide. In some embodiments,the riboswitch can regulate the inclusion of a poly(A) tail and belocated after the target polynucleotide. In some embodiments, theriboswitch can regulate an anti-IRES and be located upstream of an IRES.In non-limiting illustrative embodiments, a riboswitch provided hereincan be located in any of these positions relative to a nucleic acidencoding one or more engineered signaling polypeptides provided herein.

In any of the embodiments disclosed herein that include a polynucleotideincluding a riboswitch, the polynucleotide can further include one ormore of a Kozak-type sequence, a WPRE element, and a double stop codonor a triple stop codon, wherein one or more of the stop codons of thedouble stop codon or the triple stop codon define a termination of areading from of at least one of the one or more transcriptional units.In certain embodiments, the polynucleotide includes a Kozak-typesequence selected from CCACCAUG(G) (SEQ ID NO:515), CCGCCAT/UG(G) (SEQID NO:516), GCCGCCGCCAT/UG(G) (SEQ ID NO:517), or GCCGCCGCCAT/UG. Incertain embodiments, both the −3 and +4 nucleotides relative to a startcodon of the first nucleic acid sequence include a G. In anotherembodiment, which can be combined with the preceding embodiment thatincludes a Kozak-type sequence and/or the following embodiment thatincludes triple stop codon, the polynucleotide includes a WPRE element.In some embodiments, the WPRE element is located 3′ of a stop codon ofthe one or more transcriptional units and 5′ to a 3′ LTR of thepolynucleotide. In another embodiment, which can be combined with eitheror both of the preceding embodiments (i.e. an embodiment thepolynucleotide includes a Kozak-type sequence and/or an embodimentwherein the polynucleotide included a WPRE), the one or moretranscriptional units terminates with one or more stop codons of adouble stop codon or a triple stop codon.

In some embodiments, the riboswitch can be destabilized at temperaturesabove 37.5° C., 38° C., 38.5° C., 39° C., 39.5° C., or 40° C. such thatthe riboswitch is no longer responsive to the ligand. In someembodiments, molecular evolution can be used to select riboswitches thatare destabilized at temperatures above 37.5° C., 38° C., 38.5° C., 39°C., 39.5° C., or 40° C.

In some embodiments, the target polynucleotide can encode a miRNA,shRNA, and/or a polypeptide, wherein the target polynucleotide isoperably linked to a promoter. In some embodiments, the targetpolynucleotide can encode a lymphoproliferative element. In someembodiments, the target polynucleotide can be an miRNA or shRNA. In someembodiments, the miRNA or shRNA can potentiate the STATS pathway orinhibit the SOCS pathway. In some embodiments, the miRNA or shRNA cantarget transcripts from SOCS1, SMAD2, TGFb, or PD-1. In someembodiments, the miRNA is miR-155. In some embodiments, the targetpolynucleotide encodes a polypeptide and the polypeptide can include aCAR including an antigen-specific targeting region, a transmembranedomain, and an intracellular activating domain.

In another aspect, provided herein is an isolated polynucleotide forregulating expression of a target polynucleotide, including: apolynucleotide encoding the target polynucleotide operably linked to apromoter and a riboswitch, wherein the riboswitch includes: a.) anaptamer domain capable of binding a nucleoside analogue antiviral drugwith a binding affinity at least two-fold greater affinity than theaptamer domain binds guanine or 2′-deoxyguanosine; and b.) a functionswitching domain capable of regulating expression of the targetpolynucleotide, wherein binding of the nucleoside analogue by theaptamer domain induces or suppresses the expression regulating activityof the function switching domain. In some embodiments, the aptamerdomain can bind the nucleoside analogue antiviral drug with a bindingaffinity at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80,90, or 100-fold greater affinity than the aptamer domain binds guanineor 2′-deoxyguanosine. In some embodiments, the aptamer domain can bebetween 30, 35, 40, 45, 50, 55, 60, 65, and 70 nucleotides in length onthe low end of the range and 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, and 100 nucleotides in length on the high end of the range, forexample between 45 and 80 nucleotides in length or between 45 and 58nucleotides in length. In illustrative embodiments, the nucleosideanalogue antiviral drug can be the pharmaceutical ligand acyclovir (alsoknown as aciclovir and acycloguanosine) or penciclovir. In someembodiments, the aptamer domain can have a binding affinity to thenucleoside analogue antiviral drug that is greater than, for example atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or100-fold greater than the binding affinity to the nucleoside ornucleotide. In some embodiments, binding of the nucleoside analogue bythe aptamer domain can induce an activity in the riboswitch.

In some embodiments, the aptamer domain can be specific for penciclovirand lack reactivity to acyclovir or alternatively another antiviralagent, such that concomitant antiviral therapy may be utilized withoutaffecting the riboswitch. In some embodiments, the aptamer domain canbind penciclovir with a binding affinity at least 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100-fold greater than theaptamer domain binds acyclovir or another antiviral agent. In someembodiments, the aptamer domain can be specific for acyclovir and lackreactivity to penciclovir or alternatively another antiviral agent, suchthat concomitant antiviral therapy may be utilized without affecting theriboswitch. In some embodiments, the aptamer domain can bind acyclovirwith a binding affinity at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,50, 60, 70, 80, 90, or 100-fold greater than the aptamer domain bindspenciclovir or another antiviral agent. In some embodiments, the oralprodrugs of penciclovir (famciclovir) and acyclovir (valaciclovir) canbe given to a subject. In some embodiments, the derived aptamer domaincan be at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% identical tothe aptamer domain from the type I-A deoxyguanosine riboswitch fromMesoplasma florum. In some embodiments, the derived aptamer domain canbe at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% identical to theaptamer domain from the xpt riboswitch from Bacillus subtilis. In any ofthe embodiments disclosed herein, the riboswitch can affect the RNAtranscript by regulating any of the following activities: internalribosomal entry site, pre-mRNA splice donor accessibility in theretroviral gene construct, pre-mRNA splicing, translation, terminationof transcription, transcript degradation, miRNA expression, or shRNAexpression. In some embodiments, the function switching domain cancontrol binding of an anti-IRES to an IRES. In any of the embodimentsdisclosed herein, the presence or absence of the small molecule ligandcan cause the riboswitch to affect the RNA transcript. In someembodiments, the riboswitch can include a ribozyme. Riboswitches withribozymes can inhibit or enhance transcript degradation of genes ofinterest in the presence of the small molecule ligand. In someembodiments, the ribozyme can be a pistol class of ribozyme, ahammerhead class of ribozyme, a twisted class of ribozyme, a hatchetclass of ribozyme, or the HDV (hepatitis delta virus) ribozyme. In someembodiments, the riboswitch can be destabilized at temperatures above37.5° C., 38° C., 38.5° C., 39° C., 39.5° C., or 40° C. such that theriboswitch is no longer responsive to the ligand. In some embodiments,molecular evolution can be used to select riboswitches that aredestabilized at temperatures above 37.5° C., 38° C., 38.5° C., 39° C.,39.5° C., or 40° C. In some embodiments, the target polynucleotide canencode a miRNA, shRNA, and/or a polypeptide, wherein the targetpolynucleotide is operably linked to a promoter. In some embodiments,the target polynucleotide can encode a lymphoproliferative element. Insome embodiments, the target polynucleotide can be an miRNA and,optionally, the miRNA can stimulate the STATS pathway or inhibit theSOCS pathway. In some embodiments, the miRNA can target transcripts fromSOCS1, SHP, SMAD2, TGFb, or PD-1. In these embodiments, the miRNA can bemiR-155. In some embodiments, the target polynucleotide encodes apolypeptide and the polypeptide can include a CAR including anantigen-specific targeting region, a transmembrane domain, and anintracellular activating domain. Further embodiments of CARs aredisclosed elsewhere herein.

In some embodiments, the evolution of aptamers can be performed viaaptamer selection from randomized native purine or guanine aptamerlibraries using SELEX (Systematic Evolution of Ligands by EXponentialenrichment) methods including, but not limited to, those methods thatemploy graphene oxide in the selection process and screening. In otherembodiments, random mutagenesis methodology such as error prone PCR canbe used to evolve aptamer constructs or riboswitch constructs where theaptamer is incorporated in the context of any of the riboswitchactivities described herein by screening in vitro or in mammalian cells.In other embodiments, random libraries of nucleotides can be used in theevolution of the riboswitch. In any of the embodiments disclosed herein,riboswitches can be identified from screening such libraries in vitro orin mammalian cells.

In some embodiments, the evolved or derived aptamer domain can haveincreased binding to analogues of the native ligand and decreasedbinding to the native ligand. In some embodiments, the aptamer domaincan be configured to have increased binding to analogues of the nativeligand and decreased binding to the native ligand. In some embodiments,the aptamer domain can be derived from the purine riboswitch family. Insome embodiments, the native ligand can be a nucleoside or nucleotideand the analogue can be a nucleoside analogue or nucleotide analogue. Insome embodiments, the nucleoside analogue is an antiviral drug. Inillustrative embodiments, the aptamer domains can be derived from2′-deoxyguanosine and guanine riboswitch scaffolds and the derivedaptamer domains can show reduced binding to 2′-deoxyguanosine andguanine relative to the wild-type riboswitch.

In some embodiments, the riboswitch can regulate pre-mRNA splice donoraccessibility in the retroviral gene construct, wherein the retroviralconstruct drives the CAR genes or other genes of interest from thereverse strand under a general promoter or a T cell specific promoter.In other embodiments, the riboswitch can regulate an IRES in theretroviral gene construct, wherein the retroviral construct drives thetranslation of CAR genes or other genes of interest. In otherembodiments, the riboswitch can control transcription termination of theRNA, miRNA, or gene transcripts or can control translation of thetranscript. In other embodiments, the nucleoside analogue riboswitch canbe integrated with a ribozyme to inhibit or enhance transcriptdegradation of the CAR genes or other genes of interest in the presenceof the nucleoside analogue.

In some embodiments, the isolated polynucleotide for regulatingexpression of a target polynucleotide that includes a polynucleotideencoding the target polynucleotide operably linked to a promoter and ariboswitch that binds a nucleoside analogue antiviral drug, is amolecular cloning vector. The molecular cloning vector can be any typeof molecular cloning vector known in the art. As non-limiting examples,the vector can be a plasmid, a virus, a replication incompetent viralparticle, a retrovirus, or a replication incompetent recombinantretroviral particle, any of which can be an expression vector. Such anexpression vector can encode any of the target polynucleotides providedhereinabove. One or more restriction and/or multiple cloning sites canbe included on a molecular cloning vector 5′ or 3′ to a riboswitchprovided herein such that the riboswitch is operably linked to a targetpolynucleotide inserted into the restriction and/or multiple cloningsite.

Molecular Chaperones

In one aspect, provided herein is a method for genetically modifying andexpanding lymphocytes of a subject, comprising:

-   -   A. contacting resting T cells and/or NK cells of the subject ex        vivo, typically without requiring prior ex vivo stimulation,        with replication incompetent recombinant retroviral particles        comprising:        -   i. a pseudotyping element on its surface that is capable of            binding to a T cell and/or NK cell and facilitating membrane            fusion of the replication incompetent recombinant retroviral            particles thereto; and        -   ii. a polynucleotide comprising one or more transcriptional            units operatively linked to a promoter active in T cells            and/or NK cells, wherein the one or more transcriptional            units encode a first engineered signaling polypeptide            regulated by a control element, wherein said first            engineered signaling polypeptide comprises at least one            lymphoproliferative element and/or a chimeric antigen            receptor, wherein said contacting facilitates transduction            of at least some of the resting T cells and/or NK cells by            the replication incompetent recombinant retroviral            particles, thereby producing genetically modified T cells            and/or NK cells;    -   B. introducing the genetically modified T cells and/or NK cells        into the subject; and    -   C. exposing the genetically modified T cells and/or NK cells in        vivo to a compound that acts as the control element to affect        expression of the first engineered signaling polypeptide and        promote expansion of the lymphocytes in vivo, thereby        genetically modifying and expanding lymphocytes of the subject.

In illustrative embodiments, the transduction is carried out without exvivo stimulation. In illustrative embodiments, the compound is amolecular chaperone, such as a small molecule molecular chaperone. Inillustrative embodiments, binding of the molecular chaperone to thelymphoproliferative element and/or CAR component increases theproliferative activity of the lymphoproliferative element and/or theCAR. The molecular chaperone can be administered to the subject beforethe blood is collected, during the contacting, and/or after the T cellsand/or NK cells are introduced into the subject. Some embodiments ofthis aspect include collecting blood from the subject. In theseembodiments, the introducing is a reintroducing of the cells that werecollected and genetically modified before reintroduction. The entireprocess, in illustrative embodiments, is a shorter process than priorart methods, as for other aspects herein. For example, the entireprocess can be completed in less than 48 hours, less than 24 hours, orless than 12 hours. The entire process in other embodiments, can becompleted in 2, 4, 6, or 8 hours on the low end of the range, and 12,24, 36, or 48 hours on the high end of the range.

Accordingly, in some embodiments of the methods and compositionsprovided herein, the control element is a molecular chaperone. Ascompared to other embodiments herein with other in vivo controlelements, such as riboswitches that typically bind a compound to affectexpression of a lymphoproliferative element or other component of afirst or second engineered signaling polypeptide herein, the molecularchaperones are compounds that are the control elements and as such,directly affect activity of, typically by binding to, alymphoproliferative element or other component of a first or secondengineered signaling polypeptide herein. In illustrative examples ofsuch embodiments of methods herein that include the administration ofmolecular chaperones, a lymphoproliferative element, membrane-boundcytokine, and/or CAR component, can be a less active or inactivelymphoproliferative element, membrane-bound cytokine, and/or CARcomponent, that is bound by the molecular chaperone to increase itsactivity. Thus, the target bound by a molecular chaperone is typically atarget polypeptide. In some embodiments, as indicated the polypeptidecan be a first and/or a second engineered signaling polypeptide, or apolypeptide component thereof, whose activity is affected by binding tothe molecular chaperone, which in illustrative embodiments is a smallmolecule molecular chaperone. In some embodiments, the polypeptide caninclude a lymphoproliferative element whose activity is regulated, inillustrative embodiments, up-regulated by a molecular chaperone,preferably a small molecule molecular chaperone. The molecular chaperonein the methods provided herein can be a compound that binds to themutant lymphoproliferative element and/or inactive CAR component, thusrendering them active.

In other embodiments, a lymphoproliferative element or other signalingdomain has been mutated to permit transit to the plasma membrane only inthe presence of a small molecular synthetic chaperone. In otherembodiments, the chaperone promotes stability of the lymphoproliferativeelement or other signaling domain or protein and half-life as apotentiator.

It will be understood that aspects and embodiments of the presentinvention include many of the same steps and compositions provided indetail herein. Accordingly, it will be understood that the teachingsthroughout this specification that relate to these common elements applyto aspects and embodiments that utilize a molecular chaperone as thecontrol element, which typically binds a lymphoproliferative element orother target molecule directly, in addition to, or instead of other invivo control elements provided herein, such as riboswitches, whichtypically utilize a molecule, such as a drug, that binds the riboswitch.

In some embodiments, the molecular chaperone is a compound that canregulate sub-cellular localization of a target, for example, the properfolding and transit of a target protein, such as a lymphoproliferativeelement and/or a component of a CAR, from the endoplasmic reticulum tothe plasma membrane or its half-life on the surface. In otherembodiments, the molecular chaperone can promote the functionalconformation of a dysfunctional target, thus acting as a potentiator.Examples of molecules that act as chaperones or potentiators tonaturally mutated proteins include lumacaftor and ivacaftor. Theseproteins act upon the mutant CFTR chloride channel variants such asG551D or F508del. Ivacaftor potentiates the activity of the G551D orF508del mutated ion channel, whereas lumacaftor promotes stabilizationof mutant chloride channels and subsequent potentiation by ivacaftor.Such chaperone dependent proteins can be generated from naturallyfunctional proteins and screening for functional activity only in thepresence of the molecular chaperones. Thus, such proteins are onlyactive when the chaperone is present. Examples of such molecules whichcan be screened for specific chaperone activity include small moleculeantivirals or anti-infectives that show no activity to normal humanproteins. Accordingly, in one embodiment, the molecular chaperone usedin methods herein is a small molecule antiviral or anti-infectivecompound that shows no activity to normal human proteins.

In some embodiments, genetically modified lymphocytes can be exposedand/or a subject can be administered the molecular chaperone. In someembodiments, the compound is administered to the subject before, during,and/or after PBLs are isolated from the blood and before T cells and/orNK cells are contacted with a replication incompetent recombinantretroviral particle. The replication incompetent recombinant retroviralparticle in such embodiments includes a less active or inactivelymphoproliferative element and/or CAR component that binds to, and isregulated by, the molecular chaperone compound.

For any of the embodiments provided herein for modifying and expandinglymphocytes, which can be part of methods of adoptive cell therapy, thecompound can be administered to the subject for between 5, 10, 15, 30,and 60 minutes on the low end of the range, and 1.5, 2, 3, 4, 5, 6, 8,12, or 24 hours on the high end of the range, before PBLs are isolatedfrom the blood or before T cells and/or NK cells are contacted with areplication incompetent recombinant retroviral particle. In someembodiments, the compound is administered to the subject for between1.5, 2, 3, 4, 5, 6, 8, 12, or 24 hours on the low end of the range, ½,1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 days on the high end of therange, after PBLs are isolated from the blood or after T cells and/or NKcells are contacted with a replication incompetent recombinantretroviral particle in methods provided herein. In some embodiments, thecompound is administered to the subject for at least 1.5, 2, 3, 4, 5, 6,8, 12, or 24 hours, or at least 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 daysafter PBLs are isolated from the blood or after T cells and/or NK cellsare contacted with a replication incompetent recombinant retroviralparticle in methods provided herein. In some embodiments, the compoundis administered to the subject for at least 1, 2, 3, 4, 5, 7, 10, 14,21, 28, 30, 60, 90, or 120 days or 5, 6, 9, 12, 24, 36, 48, 60, 72, 84,96, 120 months or indefinitely after the PBLs have been reinfused intothe subject. In any of the embodiments disclosed herein, the compoundcan be administered before and/or during the reinfusion of the PBLsand/or after the PBLs have been reinfused.

For any of the embodiments herein, molecular chaperones are not in thecontrol elements that are bound by compounds that regulate and/oractivate them. Molecular chaperones are compounds, preferably smallmolecule compounds, that are the control elements and regulate theactivity of lymphoproliferative elements and/or functional components ofCARs.

Packaging Cell Lines/Methods of Making Recombinant Retroviral Particles

The present disclosure provides mammalian packaging cells and packagingcell lines that produce replication incompetent recombinant retroviralparticles. The cell lines that produce replication incompetentrecombinant retroviral particles are also referred to herein aspackaging cell lines. A non-limiting example of such method is providedin Example 7 herein. The cells of the packaging cell line can beadherent or suspension cells. In illustrative embodiments, the packagingcell line can be a suspension cell line, i.e. a cell line that does notadhere to a surface during growth. The cells can be grown in achemically-defined media and/or a serum-free media. In some embodiments,the packaging cell line can be a suspension cell line derived from anadherent cell line, for example, the HEK293 cell line can be grown inconditions to generate a suspension-adapted HEK293 cell line accordingto methods known in the art. The packaging cell line is typically grownin a chemically defined media. In some embodiments, the packaging cellline media can include serum. In some embodiments, the packaging cellline media can include a serum replacement, as known in the art. Inillustrative embodiments, the packaging cell line media can beserum-free media. Such media can be a chemically defined, serum-freeformulation manufactured in compliance with Current Good ManufacturingPractice (CGMP) regulations of the US Food and Drug Administration(FDA). The packaging cell line media can be xeno-free and complete. Insome embodiments, the packaging cell line media has been cleared byregulatory agencies for use in ex vivo cell processing, such as an FDA510(k) cleared device. It will be understood herein where it is recitedthat the media includes the composition of the basal media with a mediasupplement of a catalog number such as A1048501 or A1048503 thatincludes a media supplement, that the recited composition is intended tomean the composition of the media with the added supplement. Typically,the manufacture of the media and supplement provides instructions forvolumes of supplement to be added.

Accordingly, in one aspect, provided herein is a method of making areplication incompetent recombinant retroviral particle including: A.culturing a packaging cell in suspension in serum-free media, whereinthe packaging cell comprises nucleic acid sequences encoding apackagable RNA genome of the replication incompetent retroviralparticle, a REV protein, a gag polypeptide, a pol polypeptide, and apseudotyping element; and B. harvesting the replication incompetentrecombinant retroviral particle from the serum-free media. In anotheraspect, provided herein is a method of transducing a lymphocyte with areplication incompetent recombinant retroviral particle comprising: A.culturing a packaging cell in suspension in serum-free media, whereinthe packaging cell comprises nucleic acid sequences encoding apackagable RNA genome of the replication incompetent retroviralparticle, a REV protein, a gag polypeptide, a pol polypeptide, and apseudotyping element; B. harvesting the replication incompetentrecombinant retroviral particle from the serum-free media; and C.contacting the lymphocyte with the replication incompetent recombinantretroviral particle, wherein the contacting is performed for less than24 hours, 20 hours, 18 hours, 12 hours, 8 hours, 4 hours or 2 hours (orbetween 1, 2, 3, or 4 hours on the low end of the range and 4, 6, 8, 12,18, 20, or 24 hours on the high end of the range), thereby transducingthe lymphocyte.

In another aspect, provided herein is a retroviral packaging systemincluding: a mammalian cell including: a) a first transactivatorexpressed from a constitutive promoter and capable of binding a firstligand and a first inducible promoter for affecting expression of anucleic acid sequence operably linked thereto in the presence versusabsence of the first ligand; b) a second transactivator capable ofbinding a second ligand and a second inducible promoter, and affectingexpression of a nucleic acid sequence operably linked thereto in thepresence versus absence of a second ligand; and c) a packageable RNAgenome for a retroviral particle, wherein the first transactivatorregulates expression of the second transactivator, and wherein thesecond transactivator regulates expression of retroviral polypeptidesinvolved in viral packaging, such as, for example, a gag polypeptide, apol polypeptide, and/or a pseudotyping element, and optionally otherpolypeptides that will become incorporated in or on the replicationincompetent recombinant retroviral particle and are believed to be toxicto packaging cell lines, such as, for example, HEK-293. In certainaspects, the second transactivator itself is cytotoxic to packaging celllines. Pseudotyping elements are typically capable of binding to a cellmembrane of a target cell and facilitating fusion thereto, as discussedin detail herein. Thus, not to be limited by theory, the system providesthe ability to accumulate certain polypeptides/proteins that do notinhibit, or do not substantially inhibit, or are not believed to inhibitproliferation or survival of the mammalian cells, for example, non-toxicproteins, while culturing a population of the mammalian cells for daysor indefinitely, and controlling induction of polypeptides that aredesired for retroviral product but that are inhibitory or can beinhibitory or have been reported to be inhibitory to the survival and/orproliferation of the mammalian cell, for example toxic polypeptides,until a later time closer to the time of when replication incompetentrecombinant retroviral particles will be produced and harvested. Thepackageable RNA genome is typically encoded by a polynucleotide operablylinked to a promoter, sometimes referred to herein as a third promoterfor convenience, wherein said third promoter is typically inducible byeither the first transactivator or the second transactivator. Inillustrative embodiments, the packageable RNA genome is encoded by apolynucleotide operably linked to a third promoter, wherein said thirdpromoter is inducible by the second transactivator. As such, thepackageable RNA genome can be produced at the later time point, closerto when the replication incompetent recombinant retroviral particleswill be harvested.

A skilled artisan will appreciate many different transactivators,ligands, and inducible promoters can be used in the retroviral packagingsystem. Such inducible promoters can be isolated and derived from manyorganisms, e.g., eukaryotes and prokaryotes. Modification of induciblepromoters derived from a first organism for use in a second organism,e.g., a first prokaryote and a second a eukaryote, a first eukaryote anda second a prokaryote, etc., is well known in the art. Such induciblepromoters, and systems based on such inducible promoters but alsoincluding additional control proteins, include, but are not limited to,alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) genepromoter, promoters responsive to alcohol transactivator proteins(AlcR), etc.), tetracycline regulated promoters, (e.g., promoter systemsincluding TetActivators, TetON, TetOFF, etc.), steroid regulatedpromoters (e.g., rat glucocorticoid receptor promoter systems, humanestrogen receptor promoter systems, retinoid promoter systems, thyroidpromoter systems, ecdysone promoter systems, mifepristone promotersystems, etc.), metal regulated promoters (e.g., metallothioneinpromoter systems, etc.), pathogenesis-related regulated promoters (e.g.,salicylic acid regulated promoters, ethylene regulated promoters,benzothiadiazole regulated promoters, etc.), temperature regulatedpromoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90,soybean heat shock promoter, etc.), light regulated promoters, syntheticinducible promoters, and the like. In some embodiments, amifepristone-regulated system can be used. In some embodiments, amifepristone-inducible system with an autoregulatory feedback loop canbe used. In some embodiments, a GAL4 regulatory fusion protein isexpressed from one construct that also contains the transposon terminalrepeats and lox and FRT sites. In some embodiments, the GAL4 regulatoryfusion protein controls expression of a reverse tet transactivator(rtTA) and BiTRE. In some embodiments, another construct with lox andFRT sites contains a GAL4 upstream activating sequences (UAS) and an E1bTATA box promoter driving a reporter like mCherry. In some embodiments,a GAL4 regulatory fusion protein binds to GAL4 upstream activatingsequences (UAS) in both the promoter controlling expression of the GAL4regulatory fusion protein and the promoter controlling expression of atarget polynucleotide. In some embodiments, mifepristone, doxycycline,and puromycin will be used for induction and selection of packaging cellline.

In some embodiments, either or both transactivators can be split intotwo or more polypeptides. In some embodiments, the two or morepolypeptides can include a DNA binding domain and an activation domaincapable of stimulating transcription on separate polypeptides. This“activation domain” is not to be confused with an “activation element,”such as a polypeptide that binds CD3, which is capable of activating a Tcell and/or NK cell, and typically does activate such T cell and/or NKcell when contacted with it, as discussed in detail herein. The separatepolypeptides can further include fusions with polypeptides capable ofdimerization through the addition of a ligand. In some embodiments, theactivation domain can be the p65 activation domain or a functionalfragment thereof. In illustrative embodiments of the packaging systemsherein, the DNA binding domain can be the DNA binding domain from ZFHD1or a functional fragment thereof. In some embodiments, one polypeptidecan be a fusion with FKBP, or functional mutants and/or fragmentsthereof, or multiple FKBPs and another polypeptide can be a fusion withthe FRB domain of mTOR, or functional mutants and/or fragments thereof,and the ligand can be rapamycin or a functional rapalog. In someembodiments, the FRB contains the mutations K2095P, T2098L, and/orW2101F. In some embodiments, the separate polypeptides can be FKBP, orfunctional fragments thereof, and CalcineurinA, or functional fragmentsthereof, and the dimerizing agent can be FK506. In some embodiments, theseparate polypeptides can be FKBP, or functional fragments thereof, andCyP-Fas, or functional fragments thereof, and the dimerizing agent canbe FKCsA. In some embodiments, the separate polypeptides can be GAI, orfunctional fragments thereof, and GID1, or functional fragments thereof,and the dimerizing agent can be gibberellin. In some embodiments, theseparate polypeptides can be Snap-tag and HaloTag, or functionalfragments thereof, and the dimerizing agent can be HaXS. In someembodiments, the separate polypeptides can include the same polypeptide.For example, the DNA binding domain and activation domain can beexpressed as fusion proteins with FKBP or GyrB and the dimerizing agentcan be FK1012 or coumermycin, respectively. In some embodiments, theinducible promoter can be the DNA sequence where the DNA binding domaintypically binds. In some embodiments, the inducible promoter can varyfrom the DNA sequence where the DNA binding domain typically binds. Insome embodiments, either transactivator can be an rtTA, the ligand canbe tetracycline or doxycycline, and the inducible promoter can be a TRE.In illustrative embodiments, the first transactivator is the p65activation domain fused to FRB and the ZFHD1 DNA binding domain fused tothree FKBP polypeptides and the first ligand is rapamycin. In furtherillustrative embodiments, the second transactivator can be an rtTA, thesecond ligand can be tetracycline or doxycycline, and the induciblepromoter can be a TRE.

In some embodiments, the first transactivator can regulate expression ofan element to control the nuclear export of transcripts containing aconsensus sequence, such as an HIV Rev and the consensus sequence can bethe Rev response element. In illustrative embodiments, the target cellis a T cell.

In some embodiments, the pseudotyping element is a retroviral envelopepolypeptide. The pseudotyping element typically includes a bindingpolypeptide and a fusogenic polypeptide for binding to and facilitatingmembrane fusion of the target cell and viral membranes, as discussed inmore detail herein. In some embodiments, the pseudotyping element is thefeline endogenous virus (RD114) envelope protein, the oncoretroviralamphotropic envelope protein, the oncoretroviral ecotropic envelopeprotein, and/or vesicular stomatitis virus envelope protein (VSV-G). Inillustrative embodiments, the pseudotyping element includes a bindingpolypeptide and a fusogenic polypeptide derived from different proteins,as discussed in further detail herein. For example, in an illustrativeembodiment, especially where the target cell is a T cell and/or NK cell,the binding polypeptide is a hemagglutinin (H) polypeptide of a MeaslesVirus (such as the Edmonston strain of the Measles Virus), or acytoplasmic domain deletion variant thereof, and the fusogenicpolypeptide other is a fusion (F) polypeptide of a Measles Virus (suchas the Edmonston strain of the Measles Virus), or a cytoplasmic domaindeletion variant thereof. In some embodiments, the fusogenic polypeptidecan include multiple elements expressed as one polypeptide. In someembodiments, the binding polypeptide and the fusogenic polypeptide canbe translated from the same transcript but from separate ribosomebinding sites, or the polypeptide is cleaved after translation using apeptide cleavage signal or a ribosomal skip sequence, as disclosedelsewhere herein, to generate the binding polypeptide and the fusogenicpolypeptide. In some embodiments, where the binding polypeptide is aMeasles Virus H polypeptide, or a cytoplasmic domain deletion thereof,and the fusogenic polypeptide is a Measles Virus F polypeptide, or acytoplasmic domain deletion thereof, translation of the F and Hpolypeptides from separate ribosome binding sites results in a higheramount of the F polypeptide as compared to the H polypeptide. In someembodiments, the ratio of the F polypeptides (or cytoplasmic domaindeletions thereof) to H polypeptides (or cytoplasmic domain deletionsthereof) is at least 2:1, at least 3:1, at least 4:1, at least 5:1, atleast 6:1, at least 7:1, or at least 8:1.

In some embodiments, the first transactivator can regulate theexpression of an activation element capable of binding to and activatinga target cell, such as a T cell or NK cell. Any of the activationelements disclosed herein can be expressed. For example, in theseembodiments, the activation element can include: a.) a membrane-boundpolypeptide capable of binding to and activating CD3; and/or b.) amembrane-bound polypeptide capable of binding to and activating CD28. Insome embodiments, the membrane-bound polypeptide capable of binding toand activating CD3 can be an anti-CD3 antibody. In further embodiments,the anti-CD3 antibody can be anti-CD3 scFvFc. In some embodiments, themembrane-bound polypeptide capable of binding to and activating CD28 isCD80, CD86, or functional fragments thereof, such as an extracellulardomain of CD80.

In some embodiments, the second transactivator can regulate theexpression of a packageable RNA genome that can include an RNA thatencodes one or more target polypeptides, including as a non-limitingexample, any of the engineered signaling polypeptides disclosed hereinand/or one or more (e.g. two or more) inhibitory RNA molecules. Itshould be noted that it is envisioned that the retroviral packagingsystem aspect, and the method of making a replication incompetentrecombinant retroviral particle aspect, are not limited to makingreplication incompetent recombinant retroviral particles fortransduction of T cell and/or NK cells, but rather for any cell typethat can be transduced by replication incompetent recombinant retroviralparticles. The packageable RNA genome, in certain illustrativeembodiments, includes is designed to express one or more targetpolypeptides, including as a non-limiting example, any of the engineeredsignaling polypeptides disclosed herein and/or one or more (e.g. two ormore) inhibitory RNA molecules in opposite orientation (e.g., encodingon the opposite strand and in the opposite orientation), from retroviralcomponents such as gag and pol. For example, the packageable RNA genomecan include from 5′ to 3′: a 5′ long terminal repeat, or activetruncated fragment thereof; a nucleic acid sequence encoding aretroviral cis-acting RNA packaging element; a nucleic acid sequenceencoding a first and optionally second target polypeptide, such as, butnot limited to, an engineered signaling polypeptide(s) in oppositeorientation, which can be driven off a promoter in this oppositeorientation with respect to the 5′ long terminal repeat and thecis-acting RNA packaging element, which in some embodiments is called a“fourth” promoter for convenience only (and sometimes referred to hereinas the promoter active in T cells and/or NK cells). which is active in atarget cell such as a T cell and/or an NK cell but in illustrativeexamples is not active in the packaging cell or is only inducibly orminimally active in the packaging cell; and a 3′ long terminal repeat,or active truncated fragment thereof. In some embodiments, thepackageable RNA genome can include a central polypurine tract(cPPT)/central termination sequence (CTS) element. In some embodiments,the retroviral cis-acting RNA packaging element can be HIV Psi. In someembodiments, the retroviral cis-acting RNA packaging element can be theRev Response Element. The engineered signaling polypeptide driven by thepromoter in the opposite orientation from the 5′ long terminal repeat,in illustrative embodiments, is one or more of the engineered signalingpolypeptides disclosed herein and can optionally express one or moreinhibitory RNA molecules as disclosed in more detail herein.

It will be understood that promoter number, such as a first, second,third, fourth, etc. promoter is for convenience only. A promoter that iscalled a “fourth” promoter should not be taken to imply that there areany additional promoters, such as first, second or third promoters,unless such other promoters are explicitly recited. It should be notedthat each of the promoters are capable of driving expression of atranscript in an appropriate cell type and such transcript forms atranscription unit.

In some embodiments, the engineered signaling polypeptide can include afirst lymphoproliferative element. Suitable lymphoproliferative elementsare disclosed in other sections herein. As a non-limiting example, thelymphoproliferative element can be expressed as a fusion with arecognition domain, such as an eTag, as disclosed herein. In someembodiments, the packageable RNA genome can further include a nucleicacid sequence encoding a second engineered polypeptide including achimeric antigen receptor, encoding any CAR embodiment provided herein.For example, the second engineered polypeptide can include a firstantigen-specific targeting region, a first transmembrane domain, and afirst intracellular activating domain. Examples of antigen-specifictargeting regions, transmembrane domains, and intracellular activatingdomains are disclosed elsewhere herein. In some embodiments where thetarget cell is a T cell, the promoter that is active in a target cell isactive in a T cell, as disclosed elsewhere herein.

In some embodiments, the engineered signaling polypeptide can include aCAR, and the nucleic acid sequence can encode any CAR embodimentprovided herein. For example, the engineered polypeptide can include afirst antigen-specific targeting region, a first transmembrane domain,and a first intracellular activating domain. Examples ofantigen-specific targeting regions, transmembrane domains, andintracellular activating domains are disclosed elsewhere herein. In someembodiments, the packageable RNA genome can further include a nucleicacid sequence encoding a second engineered polypeptide. In someembodiments, the second engineered polypeptide can be alymphoproliferative element. In some embodiments where the target cellis a T cell or NK cell, the promoter that is active in a target cell isactive in a T cell or NK cell, as disclosed elsewhere herein.

In some embodiments, the packageable RNA genome can further include ariboswitch, as discussed in other sections herein. In some embodiments,the nucleic acid sequence encoding the engineered signaling polypeptidecan be in reverse orientation. In further embodiments, the packageableRNA genome can further include a riboswitch and, optionally, theriboswitch can be in reverse orientation. In any of the embodimentsdisclosed herein, a polynucleotide including any of the elements caninclude a primer binding site. In illustrative embodiments,transcription blockers or polyA sequences can be placed near genes toprevent or reduce unregulated transcription. In any of the embodimentsdisclosed herein, a nucleic acid sequence encoding Vpx can be on thesecond or an optional third transcriptional unit, or on an additionaltranscriptional unit that is operably linked to the first induciblepromoter.

Provided in another aspect herein is a mammalian packaging cell linecomprising a packageable RNA genome for a replication incompetentretroviral particle, wherein said packageable RNA genome comprises:

-   -   a. a 5′ long terminal repeat, or active fragment thereof;    -   b. a nucleic acid sequence encoding a retroviral cis-acting RNA        packaging element;    -   c. a polynucleotide comprising one or more nucleic acid        sequences operatively linked to a promoter active in T cells        and/or NK cells, wherein a first nucleic acid sequence of the        one or more nucleic acids encodes one or more (e.g. two or more)        inhibitory RNA molecules directed against one or more RNA        targets and a second nucleic acid sequence of the one or more        nucleic acid sequences encodes a chimeric antigen receptor (CAR)        comprising an antigen-specific targeting region (ASTR), a        transmembrane domain, and an intracellular activating domain;        and    -   d. a 3′ long terminal repeat, or active fragment thereof.

The inhibitory RNA molecules in the above aspect can include any of theinhibitory RNA molecules, as non-limiting examples, shRNA or miRNA,provided herein in other sections of this disclosure.

In some embodiments of the mammalian packaging cell line aspect, thepolynucleotide of (c) can be in reverse orientation to the nucleic acidsequence encoding the retroviral cis-acting RNA packaging element (b),the 5′ long terminal repeat (a), and/or the 3′ long terminal repeat (d).

In some embodiments of the mammalian packaging cell line aspect,expression of the packageable RNA genome is driven by an induciblepromoter active in the mammalian packaging cell line.

The promoter active in T cells and/or NK cells that drives expression ofthe inducible RNA and the CAR in these aspects provided immediatelyabove, in illustrative embodiments is not active or is only minimally orinducibly active in the packaging cell line. This promoter active in Tcells and/or NK cells in illustrative embodiments is located on thepackageable RNA genome between the nucleic acids encoding the one (e.g.two) or more inducible RNAs and the CAR and the 3′ LTR.

In any of the aspects directed to packageable cells or cell linesherein, that encode one or more inhibitory RNA molecules directedagainst one or more RNA targets, at least one and in some embodimentsall inhibitory RNA molecules can include a 5′ strand and a 3′ strandthat are partially or fully complementary to one another, wherein said5′ strand and said 3′ strand are capable of forming an 18-25 nucleotideRNA duplex. In some embodiments, the 5′ strand can be 18, 19, 20, 21,22, 23, 24, or 25 nucleotides in length, and the 3′ strand can be 18,19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In someembodiments, the 5′ strand and the 3′ strand can be the same ordifferent lengths. In some embodiments, the RNA duplex can include oneor more mismatches. In alternate embodiments, the RNA duplex has nomismatches.

In any of the aspects provided immediately above directed to packageablecells or cell lines herein, that encode inhibitory RNA moleculesdirected against one or more RNA targets, the inhibitory RNA moleculecan be a miRNA or an shRNA. In some embodiments, the inhibitory moleculecan be a precursor of a miRNA, such as for example, a Pri-miRNA or aPre-miRNA, or a precursor of an shRNA. In some embodiments, the one ormore inhibitory RNA molecules can be an artificially derived miRNA orshRNA. In other embodiments, the inhibitory RNA molecule can be a dsRNA(either transcribed or artificially introduced) that is processed intoan siRNA or the siRNA itself. In some embodiments, the inhibitory RNAmolecule can be a miRNA or shRNA that has a sequence that is not foundin nature, or has at least one functional segment that is not found innature, or has a combination of functional segments that are not foundin nature. In illustrative embodiments, at least one or all of theinhibitory RNA molecules are miR-155.

In any of the aspects provided immediately above directed to packageablecells or cell lines herein, that encode inhibitory RNA moleculesdirected against one or more RNA targets, the one or more inhibitory RNAmolecule(s), in some embodiments, can comprises from 5′ to 3′orientation: a 5′ arm, a 5′ stem, a loop, a 3′ stem that is partially orfully complementary to said 5′ stem, and a 3′ arm. In some embodiments,at least one of the two or more inhibitory RNA molecules has thisarrangement. In other embodiments, all of the two or more inhibitory RNAmolecules have this arrangement. In some embodiments, the 5′ stem can be18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In someembodiments, the 3′ stem can be 18, 19, 20, 21, 22, 23, 24, or 25nucleotides in length. In some embodiments, the loop can be 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40nucleotides in length. In some embodiments, the 5′ arm, 3′ arm, or both,are derived from a naturally occurring miRNA. In some embodiments, the5′ arm, 3′ arm, or both, are derived from a naturally occurring miRNA isselected from the group consisting of: miR-155, miR-30, miR-17-92,miR-122, and miR-21. In illustrative embodiments, the 5′ arm, 3′ arm, orboth are derived from miR-155. In some embodiments, the 5′ arm, 3′ arm,or both are derived from Mus musculus miR-155 or Homo sapiens miR-155.In some embodiments, the 5′ arm has the sequence set forth in SEQ IDNO:256 or is a functional variant thereof, such as, for example, asequence that is the same length as SEQ ID NO:256, or 95%, 90%, 85%,80%, 75%, or 50% as long as SEQ ID NO: 256 or is 100 nucleotides orless, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides orless, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides orless, 65 nucleotides or less, 60 nucleotides or less, 55 nucleotides orless, 50 nucleotides or less, 45 nucleotides or less, 40 nucleotides orless, 35 nucleotides or less, 30 nucleotides or less, or 25 nucleotidesor less; and is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or95% identical to SEQ ID NO:256. In some embodiments, the 3′ arm has thesequence set forth in SEQ ID NO:260 or is a functional variant thereof,such as, for example, the same length as SEQ ID NO:260, or 95%, 90%,85%, 80%, 75%, or 50% as long as SEQ ID NO: 260 or is a sequence that is100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less,85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less,70 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less,55 nucleotides or less, 50 nucleotides or less, 45 nucleotides or less,40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less,or 25 nucleotides or less; and is at least 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% identical to SEQ ID NO:260. In some embodiments,the 3′ arm comprises nucleotides 221-283 of the Mus musculus BIC.

In another aspect, provided herein is a method for making replicationincompetent recombinant retroviral particles, including: culturing apopulation of packaging cells to accumulate a first transactivator,wherein the packaging cells include the first transactivator expressedfrom a constitutive promoter, wherein the first transactivator iscapable of binding a first ligand and a first inducible promoter foraffecting expression of a nucleic acid sequence operably linked theretoin the presence versus absence of the first ligand, and whereinexpression of a second transactivator is regulated by the firsttransactivator; incubating the population of packaging cells includingaccumulated first transactivator in the presence of the first ligand toaccumulate the second transactivator, wherein the second transactivatoris capable of binding a second ligand and a second inducible promoterfor affecting expression of a nucleic acid sequence operably linkedthereto in the presence versus absence of the second ligand; andincubating the population of packaging cells including accumulatedsecond transactivator in the presence of the second ligand therebyinducing expression of retroviral polypeptides involved in viralpackaging, such as, for example, a gag polypeptide, a pol polypeptide,and/or a pseudotyping element, and optionally other polypeptides thatare believed to inhibit mammalian cell proliferation or survival thatwill become incorporated in or on the replication incompetentrecombinant retroviral particle, thereby making the replicationincompetent recombinant retroviral particle. In illustrativeembodiments, a packageable RNA genome is encoded by a polynucleotideoperably linked to a promoter, sometimes referred to for convenience asa “third” promoter wherein said third promoter is either constitutivelyactive or inducible by either the first transactivator or, inillustrative embodiments, the second transactivator, thereby making thereplication incompetent recombinant retroviral particle. Thepseudotyping elements are typically capable of binding to a cellmembrane of a target cell and facilitating fusion of the target cellmembrane to the replication incompetent recombinant retroviral particlemembrane. The pseudotyping elements can be any envelope proteins knownin the art. In some embodiments, the envelope protein can be vesicularstomatitis virus envelope protein (VSV-G), feline endogenous virus(RD114) envelope protein, oncoretroviral amphotropic envelope protein,and/or oncoretroviral ecotropic envelope protein. A skilled artisan willappreciate many different transactivators, ligands, and induciblepromoters can be used in the method for making a replication incompetentrecombinant retroviral particle. Suitable transactivators, ligands, andinducible promoters are disclosed elsewhere herein, including above. Askilled artisan will further appreciate that the teachings hereinaboverelated to a retroviral packaging system aspect provided herein, applyto method of making replication incompetent recombinant retroviralparticles aspects as well, and the reverse.

In some embodiments, the first transactivator can regulate expression ofan element to control the nuclear export of transcripts containing aconsensus sequence, such as an HIV Rev and the consensus sequence can bethe Rev Response Element (RRE). In illustrative embodiments, the targetcell is typically a T cell. In some embodiments, the HIV RREs and thepolynucleotide region encoding HIV Rev can be replaced with HIV-2 RREsand a polynucleotide region encoding the HIV-2 Rev, respectively. Insome embodiments, the HIV RREs and the polynucleotide region encodingHIV Rev can be replaced with SIV RREs and a polynucleotide regionencoding the SIV Rev, respectively. In some embodiments, the HIV RREsand the polynucleotide region encoding HIV Rev can be replaced withRemREs and a polynucleotide region encoding a betaretrovirus Rem,respectively. In some embodiments, the HIV RREs and the polynucleotideregion encoding HIV Rev can be replaced with a deltaretrovirus RexRREand a polynucleotide region encoding a deltaretrovirus Rex,respectively. In some embodiments, a Rev-like protein is not requiredand the RREs can be replaced with cis-acting RNA elements, such as theconstitutive transport element (CTE).

In some embodiments, the pseudotyping element is a viral envelopeprotein. The pseudotyping element typically includes a bindingpolypeptide and a fusogenic polypeptide for binding to and facilitatingmembrane fusion of viral and target cell membranes. In some embodiments,the pseudotyping element can be the feline endogenous virus (RD114)envelope protein, the oncoretroviral amphotropic envelope protein, theoncoretroviral ecotropic envelope protein, and/or vesicular stomatitisvirus envelope protein (VSV-G). In illustrative embodiments, thepseudotyping element includes a binding polypeptide and a fusogenicpolypeptide derived from different proteins, as discussed in furtherdetail herein. For example, in an illustrative embodiment, especiallywhere the target cell is a T cell and/or NK cell, the bindingpolypeptide can be a cytoplasmic domain deletion variant of a MeaslesVirus H polypeptide and the fusogenic polypeptide can be the cytoplasmicdomain deletion variant of a Measles Virus F polypeptide. In someembodiments, the fusogenic polypeptide can include multiple elementsexpressed as one polypeptide. In some embodiments, the bindingpolypeptide and fusogenic polypeptide can be translated from the sametranscript and translated from separate ribosome binding sites, or thepolypeptide can be cleaved after translation using a peptide cleavagesignal or a ribosomal skip sequence, as disclosed elsewhere herein, togenerate the binding polypeptide and the fusogenic polypeptide. In someembodiments, the translation of the binding polypeptide and fusogenicpolypeptide from separate ribosome binding sites results in a higheramount of the fusogenic polypeptide as compared to the bindingpolypeptide. In some embodiments, the ratio of the fusogenic polypeptideto the binding polypeptide is at least 2:1, at least 3:1, at least 4:1,at least 5:1, at least 6:1, at least 7:1, or at least 8:1.

In some embodiments, the first transactivator can regulate theexpression of an activation element capable of binding to and activatinga target cell, such as a T cell. In these embodiments, the activationelement can include: a.) aa membrane-bound polypeptide capable ofbinding to and activating CD3: and/or b.) a membrane-bound polypeptidecapable of binding to and activating CD28. In some embodiments, themembrane-bound polypeptide capable of binding to and activating CD28 isCD80, CD86, or functional fragments thereof. In some embodiments, thereplication incompetent recombinant retroviral particle can include theactivation element on a retroviral membrane and the retroviral RNAwithin a nucleocapsid, thereby making a replication incompetentrecombinant retroviral particles.

In some embodiments, the second transactivator can regulate theexpression of an RNA including from 5′ to 3′: a 5′ long terminal repeat,or active truncated fragment thereof; a nucleic acid sequence encoding aretroviral cis-acting RNA packaging element; a nucleic acid sequenceencoding a first target polypeptide and optional second targetpolypeptide, as non-limiting example, one or two engineered signalingpolypeptides; a promoter that is active in a target cell; and a 3′ longterminal repeat, or active truncated fragment thereof. In someembodiments, the RNA can include a cPPT/CTS element. In someembodiments, the RNA can include a primer binding site. In someembodiments, the retroviral cis-acting RNA packaging element can be HIVPsi. In some embodiments, the retroviral cis-acting RNA packagingelement can be the Rev Response Element. In any of the embodimentsdisclosed herein, retroviral components on the RNA, including RRE andPsi, can be located in any position, as a skilled artisan willunderstand. The engineered signaling polypeptide in illustrativeembodiments, is one or more of the engineered signaling polypeptidesdisclosed herein.

In some embodiments, the engineered signaling polypeptide can include afirst lymphoproliferative element. Suitable lymphoproliferative elementsare disclosed in other sections herein. In some illustrativeembodiments, the lymphoproliferative element is an IL-7 receptor mutantfused to a recognition domain, such as an eTag. In some embodiments, thepackageable RNA genome can further include a nucleic acid sequenceencoding a second engineered polypeptide including a chimeric antigenreceptor, encoding any CAR embodiment provided herein. For example, thesecond engineered polypeptide can include a first antigen-specifictargeting region, a first transmembrane domain, and a firstintracellular activating domain. Examples of antigen-specific targetingregions, transmembrane domains, and intracellular activating domains aredisclosed elsewhere herein. In some embodiments where the target cell isa T cell, the promoter that is active in a target cell is active in a Tcell, as disclosed elsewhere herein.

In some embodiments, the packageable RNA genome can further include ariboswitch, as discussed in other sections herein. In some embodiments,the nucleic acid sequence encoding the engineered signaling polypeptidecan be in reverse orientation. In further embodiments, the packageableRNA genome can further include a riboswitch and, optionally, theriboswitch can be in reverse orientation. In any of the embodimentsdisclosed herein, a polynucleotide including any of the elements caninclude a primer binding site. In illustrative embodiments,transcription blockers or polyA sequences can be placed near genes toprevent or reduce unregulated transcription. In any of the embodimentsdisclosed herein, a nucleic acid sequence encoding Vpx can be on thesecond or an optional third transcriptional unit, or on an additionaltranscriptional unit that is operably linked to the first induciblepromoter.

In some embodiments of the packaging system or methods for makingreplication incompetent recombinant retroviral particles aspects, theencoded RNA can include an intron, which can be transcribed, forexample, from the same promoter for expressing the targetpolypeptide(s). Such intron can encode 1, 2, 3, or 4 miRNAs, in certainillustrative embodiments. In these and other embodiments of thepackaging system or methods for making replication incompetentrecombinant retroviral particles aspects, the packageable RNA genome is11,000 KB or less and in some instances 10,000 KB or less in size.

In some embodiments, the first transactivator can affect the expressionof one or more polypeptides that are non-toxic. In some embodiments, thesecond transactivator can affect the expression of one or morepolypeptides that are toxic. For example, the first transactivator caninduce expression of the retroviral proteins Rev and Vpx in addition topolypeptides that will be transported to the cell membrane of thepackaging cell and the second transactivator can induce expression ofthe retroviral proteins GAG, POL, MV(Ed)-FΔ30, and either MV(Ed)-HΔ18 orMV(Ed)-HΔ24 and expression of the lentiviral genome. In someembodiments, the first transactivator can affect the expression of oneor more polypeptides that are toxic and/or the second transactivator canaffect the expression of one or more polypeptides that are non-toxic.

In another aspect, provided herein is a mammalian packaging cell,including: a.) a first transcriptional unit in the genome of themammalian packaging cell, including a nucleic acid sequence encoding afirst transactivator, wherein said first transcriptional unit isoperably linked to a constitutive promoter and wherein saidtransactivator is capable of binding a first inducible promoter andaffecting expression of a nucleic acid sequence operably linked theretoin the presence versus absence of a first ligand, and wherein said firsttransactivator is capable of binding said first ligand; b.) a second andoptional third transcriptional unit in the genome of the mammalianpackaging cell, including a nucleic acid sequence encoding a retroviralREV protein and a nucleic acid sequence encoding a second transactivatorcapable of binding a second inducible promoter and affecting expressionof a nucleic acid sequence operably linked thereto in the presenceversus absence of a second ligand, wherein the second transactivator iscapable of binding the second ligand, and wherein the second andoptional third transcriptional units are operably linked to the firstinducible promoter; c.) a fourth and optional fifth transcriptional unitin the genome of the mammalian packaging cell, including a nucleic acidsequence encoding a retroviral gag polypeptide and a retroviral polpolypeptide, and a binding polypeptide and a fusogenic polypeptide thatare capable of binding to and facilitating fusion of a target cellmembrane and the retroviral membrane, wherein the fourth and optionalfifth transcriptional unit are operably linked to the second induciblepromoter; and d) a sixth transcriptional unit in the genome of themammalian packaging cell, including, from 5′ to 3′, a 5′ LTR, or activetruncated fragment thereof, a nucleic acid sequence encoding aretroviral cis-acting RNA packaging element, a cPPT/CTS element, areverse complement of a nucleic acid sequence encoding an engineeredsignaling polypeptide, an intron, a promoter that is active in a targetcell, and a 3′ LTR, or active truncated fragment thereof, wherein thesixth transcriptional unit is operably linked to the second induciblepromoter.

In another aspect, provided herein is a method for making a replicationincompetent recombinant retroviral particle, including: 1.) culturing apopulation of packaging cells to accumulate a first transactivator,wherein the packaging cells include: a.) a first transcriptional unit inthe genome of the mammalian packaging cell, including a nucleic acidsequence encoding a first transactivator, wherein said firsttranscriptional unit is operably linked to a constitutive promoter andwherein said transactivator is capable of binding a first induciblepromoter and affecting expression of a nucleic acid sequence operablylinked thereto in the presence versus absence of a first ligand, andwherein said first transactivator is capable of binding said firstligand; b.) a second and optional third transcriptional unit in thegenome of the mammalian packaging cell, including a nucleic acidsequence encoding a retroviral REV protein and a nucleic acid sequenceencoding a second transactivator capable of binding a second induciblepromoter and affecting expression of a nucleic acid sequence operablylinked thereto in the presence versus absence of a second ligand,wherein the second transactivator is capable of binding the secondligand, and wherein the second and optional third transcriptional unitsare operably linked to the first inducible promoter; c.) a fourth andoptional fifth transcriptional unit in the genome of the mammalianpackaging cell, including a nucleic acid sequence encoding a retroviralgag polypeptide and a retroviral pol polypeptide, and a bindingpolypeptide and a fusogenic polypeptide that are capable of binding toand facilitating fusion of the retroviral membrane with a target cellmembrane, wherein the fourth and optional fifth transcriptional unit areoperably linked to the second inducible promoter; and d.) a sixthtranscriptional unit in the genome of the mammalian packaging cell,including from 5′ to 3′, a 5′ LTR, or active truncated fragment thereof,a primer binding site (PBS), a nucleic acid sequence encoding aretroviral cis-acting RNA packaging element, a cPPT/CTS element, areverse complement of a nucleic acid sequence encoding an engineeredsignaling polypeptide, an intron, a target cell promoter that is activein a target cell, a 3′ LTR, or active truncated fragment thereof,wherein the fifth transcriptional unit is operably linked to the secondinducible promoter; and 2.) incubating the population of packaging cellsincluding the first transactivator in the presence of the first ligandto accumulate the second transactivator and the retroviral REV protein;and 3.) incubating the population of packaging cells including thesecond transactivator and the retroviral REV protein in the presence ofthe second ligand thereby inducing expression of the retroviral gagpolypeptide, the retroviral pol polypeptide, the binding polypeptide,the fusogenic polypeptide, and a retroviral RNA including from 5′ to 3′,a 5′ LTR, or active fragment thereof, the PBS, the retroviral cis-actingRNA packaging element, the reverse complement of the nucleic acidsequence encoding the engineered signaling polypeptide, the target cellpromoter, and a 3′ LTR, or active truncated fragment thereof, whereinreplication incompetent recombinant retroviral particles are formed andrelease from the packaging cells, and wherein the replicationincompetent recombinant retroviral particles include the bindingpolypeptide and/or the fusogenic polypeptide on a retroviral membraneand the retroviral RNA within a nucleocapsid, thereby making replicationincompetent recombinant retroviral particles.

In one aspect provided herein, the retroviral packaging system caninclude a mammalian cell including: 1.) a first transactivator expressedfrom a constitutive promoter and capable of binding a first ligand and afirst inducible promoter for affecting expression of a nucleic acidsequence operably linked thereto in the presence versus absence of thefirst ligand; 2.) a second transactivator capable of binding a secondligand and a second inducible promoter and affecting expression of anucleic acid sequence operably linked thereto in the presence versusabsence of a second ligand; and 3.) a packageable RNA genome for aretroviral particle, wherein the first transactivator regulatesexpression of the second transactivator, HIV REV, an IL7 GPI DAF, and anactivation element, and wherein the second transactivator regulatesexpression of a gag polypeptide, a pol polypeptide, a retroviralcis-acting RNA packaging element, and one or more envelope polypeptides.In illustrative embodiments, the first transactivator can be an FRBdomain fused to a p65 activation domain and one or more FKBP domainsfused to a ZFHD1 DNA binding domain, the first ligand can be rapamycin,and the first inducible promoter can be one or more ZFHD1 binding sites.In illustrative embodiments, the second transactivator can be an rtTAprotein, the second ligand can be tetracycline or doxycycline, and thesecond inducible promoter can be a TRE promoter or a bi-directional TREpromoter. In illustrative embodiments, the retroviral cis-acting RNApackaging element can be HIV Psi. In illustrative embodiments, the oneor more envelope proteins include the cytoplasmic domain deletionvariants of F and H polypeptides of a Measles Virus. In illustrativeembodiments, transcription blockers or polyA sequences can be placednear genes to prevent or reduce unregulated transcription. In someembodiments, a rapamycin-doxycycline inducible lentiviral genome withriboswitch can be used (SEQ ID NO:83). In some embodiments, arapamycin-doxycycline inducible GAG POL ENV can be used (SEQ ID NO:84).In some embodiments, a rapamycin-inducible TET activator can be used(SEQ ID NO:85). In some embodiments, a rapamycin inducer inducible REVsrcVpx can be used (SEQ ID NO:86).

Some aspects of the present disclosure include or are cells, inillustrative examples, mammalian cells, that are used as packaging cellsto make replication incompetent recombinant retroviral particles, suchas lentiviruses, for transduction of T cells and/or NK cells. Any of awide variety of cells can be selected for in vitro production of a virusor virus particle, such as a redirected recombinant retroviral particle,according to the invention. Eukaryotic cells are typically used,particularly mammalian cells including human, simian, canine, feline,equine and rodent cells. In illustrative examples, the cells are humancells. In further illustrative embodiments, the cells reproduceindefinitely, and are therefore immortal. Examples of cells that can beadvantageously used in the present invention include NIH 3T3 cells, COScells, Madin-Darby canine kidney cells, human embryonic 293T cells andany cells derived from such cells, such as gpnlslacZ pNX cells, whichare derived from 293T cells. Highly transfectable cells, such as humanembryonic kidney 293T cells, can be used. By “highly transfectable” itis meant that at least about 50%, more preferably at least about 70% andmost preferably at least about 80% of the cells can express the genes ofthe introduced DNA.

Suitable mammalian cells include primary cells and immortalized celllines. Suitable mammalian cell lines include human cell lines, non-humanprimate cell lines, rodent (e.g., mouse, rat) cell lines, and the like.Suitable mammalian cell lines include, but are not limited to, HeLacells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHOcells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCCNo. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658),Huh-7 cells, BHK cells (e.g., ATCC No. CCLlO), PC12 cells (ATCC No.CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATl cells, mouse Lcells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No.CRL1573), HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g.,NKL, NK92, and YTS), and the like.

In any of the embodiments disclosed herein, the methods of making areplication incompetent recombinant retroviral particle can includegrowing a mammalian packaging cells to 50%, 60%, 70%, 80%, 90% or 95%confluence or confluence or to 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or95% peak cell density or peak cell density and then splitting ordiluting the cells. In some embodiments, a stirred tank reactor can beused to grow the cells. In some embodiments, the cells can be split atleast about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, or1:20 using methods a skilled artisan will understand. In someembodiments, the cells can be diluted to 25%, 30%, 40%, 50%, 60%, 70%,80%, 90% or 95% peak cell density. In some embodiments, after splittingor diluting the cells the cells can be grown for 1, 2, 3, 4, 5, 6, 7, 8,10, or 16 hours or 1, 2, 3, 4, 5, 6, or 7 days before adding the firstligand. In some embodiments, the cells are grown in the presence of thefirst ligand for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, or28 days in the presence of the first ligand, which in illustrativeembodiments can be rapamycin or a rapalog. In some embodiments, thesecond ligand can be added and the cells can be grown for at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, or 28 days which inillustrative embodiments can be tetracycline or doxycyline. Conditionsfor culturing will depend on the cells and ligands used and the methodsare known in the art. A specific example of conditions for culturing andinducing HEK293S cells is shown in Example 8.

As disclosed herein, replication incompetent recombinant retroviralparticles are a common tool for gene delivery (Miller, Nature (1992)357:455-460). The ability of replication incompetent recombinantretroviral particles to deliver an unrearranged nucleic acid sequenceinto a broad range of rodent, primate and human somatic cells makesreplication incompetent recombinant retroviral particles well suited fortransferring genes to a cell. In some embodiments, the replicationincompetent recombinant retroviral particles can be derived from theAlpharetrovirus genus, the Betaretrovirus genus, the Gammaretrovirusgenus, the Deltaretrovirus genus, the Epsilonretrovirus genus, theLentivirus genus, or the Spumavirus genus. There are many retrovirusessuitable for use in the methods disclosed herein. For example, murineleukemia virus (MLV), human immunodeficiency virus (HIV), equineinfectious anaemia virus (EIAV), mouse mammary tumor virus (MMTV), Roussarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murineleukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV),Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus(A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avianerythroblastosis virus (AEV) can be used. A detailed list ofretroviruses may be found in Coffin et al (“Retroviruses” 1997 ColdSpring Harbor Laboratory Press Eds: J M Coffin, S M Hughes, H E Varmuspp 758-763). Details on the genomic structure of some retroviruses maybe found in the art. By way of example, details on HIV may be found fromthe NCBI Genbank (i.e. Genome Accession No. AF033819).

In illustrative embodiments, the replication incompetent recombinantretroviral particles can be derived from the Lentivirus genus. In someembodiments, the replication incompetent recombinant retroviralparticles can be derived from HIV, SIV, or FIV. In further illustrativeembodiments, the replication incompetent recombinant retroviralparticles can be derived from the human immunodeficiency virus (HIV) inthe Lentivirus genus. Lentiviruses are complex retroviruses which, inaddition to the common retroviral genes gag, pol and env, contain othergenes with regulatory or structural function. The higher complexityenables the lentivirus to modulate the life cycle thereof, as in thecourse of latent infection. A typical lentivirus is the humanimmunodeficiency virus (HIV), the etiologic agent of AIDS. In vivo, HIVcan infect terminally differentiated cells that rarely divide, such aslymphocytes and macrophages.

In illustrative embodiments, replication incompetent recombinantretroviral particles provided herein contain Vpx polypeptide. Vpxpolypeptide can be expressed in a packaging cell line, after integrationof a Vpx coding nucleic acid in its genome, for example as a cellmembrane bound protein that gets incorporated into a retrovirus membrane(Durand et al., J. Virol. (2013) 87: 234-242). A retroviral membranebound Vpx can be constructed with a processing sequence for a viralprotease such that free Vpx is released once incorporated in a viralparticle. Such an example of a Vpx fusion with this functionality isSrc-Flag-Vpx, which includes a membrane-targeting domain (MGSSKSKPKDP)(SEQ ID NO:227) of the first 11 amino acids of c-Src followed by a viralprotease cleavage domain KARVLAEA (SEQ ID NO:228) followed byFlag-tagged Vpx.

Not to be limited by theory, Vpx polypeptide aids in transduction ofresting cells by stimulating the efficiency of the process of reversetranscription by degrading the restriction factor SAMHD1. Accordingly,it is believed that in the methods provided herein where Vpx is presentin a replication incompetent recombinant retroviral particles used totransduce T cells and/or NK cells, Vpx is released into the cytoplasm ofa resting T cell or a resting NK cell upon transduction of the cell by areplication incompetent recombinant retroviral particle that containsVpx. Vpx then degrades SAMHD1, which causes an increase in free dNTPs,which in turn, stimulates reverse transcription of the retroviralgenome.

In some embodiments, replication incompetent recombinant retroviralparticles provided herein comprise and/or contain Vpu polypeptide. Vpupolypeptide can be expressed from a plasmid or in a packaging cell line,after integration of a Vpu coding nucleic acid in its genome, forexample, as a viral membrane protein that gets incorporated into theretrovirus membrane. A recombinant form of Vpu, with its sequence codonoptimized for expression in humans, can be constructed and expressedsuch that free Vpu can incorporate into the viral particles. Vpu is anaccessory protein found in HIV-1 and some HIV-1-related simianimmunodeficiency virus (SIV) isolates, such as SIVcpz, SIVgsn, andSIVmon, but not in HIV-2 or the majority of SIV isolates. The Vpuprotein participates in the downregulation of CD4, and antagonism oftetherin for particle release. More importantly, Vpu shares structuralsimilarities with viroporins, and particularly the viroporin protein M2from Influenza A virus (IAV), As such, Vpu has been shown to formcation-selective ion channels and permeabilize membranes, in variousmodels. It has been shown that IAV-M2 helps acidify the particles andthus favors an early release from the endosomal pathway used for entry,reducing the amount of dead-end transit products.

Example 20 herein demonstrates that the transduction of resting PBMCs byretroviral particles is enhanced by the presence of Vpu in theretroviral particles. Not to be limited by theory, Vpu polypeptide aidsin transduction of resting cells by accelerating the acidification ofthe lentiviral pseudotypes, allowing more capsids to reach the cytoplasmfaster. Acidification of virus interior, leads to weakening ofelectrostatic interaction between viral structural proteins and thusfacilitates dissociation of the capsid and viral ribonucleoprotein (RNP)complexes. Accordingly, it is believed that in the methods providedherein where Vpu is present in a replication incompetent recombinantretroviral particle membrane used to transduce T cells and/or NK cells,Vpu accelerates the acidification of the lentiviral particles in theendosomal pathway, and favors the release of capsids into the cytoplasmof resting T cells or resting NK cells upon transduction of the cells bya replication incompetent recombinant retroviral particle that containsVpu. Accordingly, in some embodiments therein a Vpu polypeptide isincluded that comprises or is a fragment of Vpu that retains the abilityto promote transduction of resting PBMCs and in some embodiments,resting T cells. The fragment can comprise a fragment of 75, 80, 85, 90,95, 96, 97, 98, 99 or 100% of Vpu that has at least 75, 80, 85, 90, 95,98, 99, or 100% identity to a wildtype Vpu. In some embodiments, a Vpupolypeptide includes a Vpu fragment that retains the ability toaccelerate acidification of a lentiviral particle in the endosomalpathway.

Retroviral Genome Size

In the methods and compositions provided herein, the recombinantretroviral genomes, in non-limiting illustrative examples, lentiviralgenomes, have a limitation to the number of polynucleotides that can bepackaged into the viral particle. In some embodiments provided herein,the polypeptides encoded by the polynucleotide encoding region can betruncations or other deletions that retain a functional activity suchthat the polynucleotide encoding region is encoded by less nucleotidesthan the polynucleotide encoding region for the wild-type polypeptide.In some embodiments, the polypeptides encoded by the polynucleotideencoding region can be fusion polypeptides that can be expressed fromone promoter. In some embodiments, the fusion polypeptide can have acleavage signal to generate two or more functional polypeptides from onefusion polypeptide and one promoter. Furthermore, some functions thatare not required after initial ex vivo transduction are not included inthe retroviral genome, but rather are present on the surface of thereplication incompetent recombinant retroviral particles via thepackaging cell membrane. These various strategies are used herein tomaximize the functional elements that are packaged within thereplication incompetent recombinant retroviral particles.

In some embodiments, the recombinant retroviral genome to be packagedcan be between 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, and8,000 nucleotides on the low end of the range and 2,000, 3,000, 4,000,5,000, 6,000, 7,000, 8,000, 9,000, 10,000, and 11,000 nucleotides on thehigh end of the range. The retroviral genome to be packaged includes oneor more polynucleotide regions encoding a first and second engineeringsignaling polypeptide as disclosed in detail herein. In someembodiments, the recombinant retroviral genome to be packaged can beless than 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, or 11,000nucleotides. Functions discussed elsewhere herein that can be packagedinclude required retroviral sequences for retroviral assembly andpackaging, such as a retroviral rev, gag, and pol coding regions, aswell as a 5′ LTR and a 3′ LTR, or an active truncated fragment thereof,a nucleic acid sequence encoding a retroviral cis-acting RNA packagingelement, and a cPPT/CTS element. Furthermore, in illustrativeembodiments a replication incompetent recombinant retroviral particleherein can include any one or more or all of the following, in someembodiments in reverse orientation of these retroviral functionalregions: one or more polynucleotide regions encoding a first and secondengineering signaling polypeptide, at least one of which includes atleast one lymphoproliferative element and can further include an ASTR; asecond engineered signaling polypeptide that can include a chimericantigen receptor; a control element, such as a riboswitch, whichtypically regulates expression of the first and/or the secondengineering signaling polypeptide; a recognition domain, an intron, apromoter that is active in a target cell, such as a T cell, a 2Acleavage signal and/or an IRES.

Recombinant Retroviral Particles

Recombinant retroviral particles are disclosed in methods andcompositions provided herein, for example, to transduce T cells and/orNK cells to make genetically modified T cells and/or NK cells. Therecombinant retroviral particles are themselves aspects of the presentinvention. Typically, the recombinant retroviral particles included inaspects provided herein, are replication incompetent, meaning that arecombinant retroviral particle cannot replicate once it leaves thepackaging cell. In illustrative embodiments, the recombinant retroviralparticles are lentiviral particles.

Provided herein in some aspects are replication incompetent recombinantretroviral particles for use in transducing cells, typically lymphocytesand illustrative embodiments T cells and/or NK cells. The replicationincompetent recombinant retroviral particles can include any of thepseudotyping elements discussed elsewhere herein. In some embodiments,the replication incompetent recombinant retroviral particles can includeany of the activation elements discussed elsewhere herein. In oneaspect, provided herein is a replication incompetent recombinantretroviral particle including a polynucleotide including: A. one or moretranscriptional units operatively linked to a promoter active in T cellsand/or NK cells, wherein the one or more transcriptional units encode achimeric antigen receptor (CAR); and B. a pseudotyping element and a Tcell activation element on its surface, wherein the T cell activationelement is not encoded by a polynucleotide in the replicationincompetent recombinant retroviral particle. In some embodiments, the Tcell activation element can be any of the activation elements discussedelsewhere herein. In illustrative embodiments, the T cell activationelement can be anti-CD3 scFvFc. In another aspect, provided herein is areplication incompetent recombinant retroviral particle, including apolynucleotide including one or more transcriptional units operativelylinked to a promoter active in T cells and/or NK cells, wherein the oneor more transcriptional units encode a first polypeptide including achimeric antigen receptor (CAR) and a second polypeptide including alymphoproliferative element. In some embodiments, thelymphoproliferative element can be a chimeric lymphoproliferativeelement. In illustrative embodiments, the lymphoproliferative elementdoes not comprise IL-7 tethered to the IL-7 receptor alpha chain or afragment thereof. In some embodiments the lymphoproliferative elementdoes not comprise IL-15 tethered to the IL-2/IL-15 receptor beta chain.

In some aspects, provided herein is a replication incompetentrecombinant retroviral particle, comprising a polynucleotide comprisingone or more transcriptional units operatively linked to a promoteractive in T cells and/or NK cells, wherein the one or moretranscriptional units encode a first polypeptide comprising a chimericantigen receptor (CAR) and a second polypeptide comprising a chimericlymphoproliferative element, for example a constitutively activechimeric lymphoproliferative element. In illustrative embodiments, thechimeric lymphoproliferative element does not comprise a cytokinetethered to its cognate receptor or tethered to a fragment of itscognate receptor.

Provided herein in some aspects, is a recombinant retroviral particlethat includes (i) a pseudotyping element capable of binding to a T celland/or NK cell and facilitating membrane fusion of the recombinantretroviral particle thereto; (ii) a polynucleotide having one or moretranscriptional units operatively linked to a promoter active in T cellsand/or NK cells, wherein the one or more transcriptional units encode afirst engineered signaling polypeptide having a chimeric antigenreceptor that includes an antigen-specific targeting region, atransmembrane domain, and an intracellular activating domain, and asecond engineered signaling polypeptide that includes at least onelymphoproliferative element; wherein expression of the first engineeredsignaling polypeptide and/or the second engineered signaling polypeptideare regulated by an in vivo control element; and (iii) an activationelement on its surface, wherein the activation element is capable ofbinding to a T cell and/or NK cell and is not encoded by apolynucleotide in the recombinant retroviral particle. In someembodiments, the promoter active in T cells and/or NK cells is notactive in the packaging cell line or is only active in the packagingcell line in an inducible manner. In any of the embodiments disclosedherein, either of the first and second engineered signaling polypeptidescan have a chimeric antigen receptor and the other engineered signalingpolypeptide can have at least one lymphoproliferative element.

Various elements and combinations of elements that are included inreplication incompetent, recombinant retroviral particles are providedthroughout this disclosure, such as, for example, pseudotyping elements,activation elements, and membrane bound cytokines, as well as nucleicacid sequences that are included in a genome of a replicationincompetent, recombinant retroviral particle such as, but not limitedto, a nucleic acid encoding a CAR; a nucleic acid encoding alymphoproliferative element; a nucleic acid encoding a control element,such as a riboswitch; a promoter, especially a promoter that isconstitutively active or inducible in a T cell; and a nucleic acidencoding an inhibitory RNA molecule. Furthermore, various aspectsprovided herein, such as methods of making recombinant retroviralparticles, methods for performing adoptive cell therapy, and methods fortransducing T cells, produce and/or include replication incompetent,recombinant retroviral particles. Replication incompetent recombinantretroviruses that are produced and/or included in such methodsthemselves form separate aspects of the present invention as replicationincompetent, recombinant retroviral particle compositions, which can bein an isolated form. Such compositions can be in dried down (e.g.lyophilized) form or can be in a suitable solution or medium known inthe art for storage and use of retroviral particles.

Accordingly, as a non-limiting example, provided herein in anotheraspect, is a replication incompetent recombinant retroviral particlehaving in its genome a polynucleotide having one or more nucleic acidsequences operatively linked to a promoter active in T cells and/or NKcells that in some instances, includes a first nucleic acid sequencethat encodes one or more (e.g. two or more) inhibitory RNA moleculesdirected against one or more RNA targets and a second nucleic acidsequence that encodes a chimeric antigen receptor, or CAR, as describedherein. In other embodiments, a third nucleic acid sequence is presentthat encodes at least one lymphoproliferative element describedpreviously herein that is not an inhibitory RNA molecule. In certainembodiments, the polynucleotide incudes one or more riboswitches aspresented herein, operably linked to the first nucleic acid sequence,the second nucleic acid sequence, and/or the third nucleic acidsequence, if present. In such a construct, expression of one or moreinhibitory RNAs, the CAR, and/or one or more lymphoproliferativeelements that are not inhibitory RNAs is controlled by the riboswitch.In some embodiments, two to 10 inhibitory RNA molecules are encoded bythe first nucleic acid sequence. In further embodiments, two to sixinhibitory RNA molecules are encoded by the first nucleic acid sequence.In illustrative embodiments, 4 inhibitory RNA molecules are encoded bythe first nucleic acid sequence. In some embodiments, the first nucleicacid sequence encodes one or more inhibitory RNA molecules and islocated within an intron. In certain embodiments, the intron includesall or a portion of a promoter. The promoter can be a Pol I, Pol II, orPol III promoter. In some illustrative embodiments, the promoter is aPol II promoter. In some embodiments, the intron is adjacent to anddownstream of the promoter active in a T cell and/or NK cell. In someembodiments, the intron is EF1-α intron A.

Recombinant retroviral particle embodiments herein include those whereinthe retroviral particle comprises a genome that includes one or morenucleic acids encoding one or more inhibitory RNA molecules. Variousalternative embodiments of such nucleic acids that encode inhibitory RNAmolecules that can be included in a genome of a retroviral particle,including combinations of such nucleic acids with other nucleic acidsthat encode a CAR or a lymphoproliferative element other than aninhibitory RNA molecule, are included for example, in the inhibitory RNAsection provided herein, as well as in various other paragraphs thatcombine these embodiments. Furthermore, various alternatives of suchreplication incompetent recombinant retroviruses can be identified byexemplary nucleic acids that are disclosed within packaging cell lineaspects disclosed herein. A skilled artisan will recognize thatdisclosure in this section of a recombinant retroviral particle thatincludes a genome that encodes one or more (e.g. two or more) inhibitoryRNA molecules, can be combined with various alternatives for suchnucleic acids encoding inhibitory RNA molecules provided in othersections herein. Furthermore, a skilled artisan will recognize that suchnucleic acids encoding one or more inhibitory RNA molecules can becombined with various other functional nucleic acid elements providedherein, as for example, disclosed in the section herein that focuses oninhibitory RNA molecules and nucleic acid encoding these molecules. Inaddition, the various embodiments of specific inhibitory RNA moleculesprovided herein in other sections can be used in recombinant retroviralparticle aspects of the present disclosure.

Necessary elements of recombinant retroviral vectors, such as lentiviralvectors, are known in the art. These elements are included in thepackaging cell line section and in details for making replicationincompetent, recombinant retroviral particles provided in the Examplessection. For example, lentiviral particles typically include packagingelements REV, GAG and POL, which can be delivered to packaging celllines via one or more packaging plasmids, a pseudotyping element,various examples which are provided herein, which can be delivered to apackaging cell line via a pseudotyping plasmid, and a genome, which isproduced by a polynucleotide that is delivered to a host cell via atransfer plasmid. This polynucleotide typically includes the viral LTRsand a psi packaging signal. The 5′ LTR can be a chimeric 5′ LTR fused toa heterologous promoter, which includes 5′ LTRs that are not dependenton Tat transactivation. The transfer plasmid can be self-inactivating,for example, by removing a U3 region of the 3′ LTR. In some non-limitingembodiments, Vpu, such as a polypeptide comprising Vpu (sometimes calleda “Vpu polypeptide” herein) including but not limited to, Src-FLAG-Vpu,is packaged within the retroviral particle for any composition or methodaspect and embodiment provided herein that includes a retroviralparticle. In some non-limiting embodiments, Vpx, such as Src-FLAG-Vpx,is packaged within the retroviral particle. Not to be limited by theory,upon transduction of a T cells, Vpx enters the cytosol of the cells andpromotes the degradation of SAMHD1, resulting in an increased pool ofcytoplasmic dNTPs available for reverse transcription. In somenon-limiting embodiments, Vpu and Vpx is packaged within the retroviralparticle for any composition or method aspect and embodiment thatincludes a retroviral particle provided herein.

Retroviral particles (e.g. lentiviral particles) included in variousaspects of the present invention are in illustrative embodiments,replication incompetent, especially for safety reasons for embodimentsthat include introducing cells transduced with such retroviral particlesinto a subject. When replication incompetent retroviral particles areused to transduce a cell, retroviral particles are not produced from thetransduced cell. Modifications to the retroviral genome are known in theart to assure that retroviral particles that include the genome arereplication incompetent. However, it will be understood that in someembodiments for any of the aspects provided herein, replicationcompetent recombinant retroviral particles can be used.

A skilled artisan will recognize that the functional elements discussedherein can be delivered to packaging cells and/or to T cells usingdifferent types of vectors, such as expression vectors. Illustrativeaspects of the invention utilize retroviral vectors, and in someparticularly illustrative embodiments lentiviral vectors. Other suitableexpression vectors can be used to achieve certain embodiments herein.Such expression vectors include, but are not limited to, viral vectors(e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus(see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994;Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:77007704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94/12649,WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655);adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86,1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., InvestOpthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al.,Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulski etal., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988)166:154-165; and Flotte et al., PNAS (1993) 90: 10613-10617); SV40;herpes simplex virus; or a retroviral vector (e.g., Murine LeukemiaVirus, spleen necrosis virus, and vectors derived from retroviruses suchas Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, humanimmunodeficiency virus, myeloproliferative sarcoma virus, and mammarytumor virus), for example a gamma retrovirus; or human immunodeficiencyvirus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi etal., J Virol 73:7812 7816, 1999); and the like.

In illustrative embodiments, a retroviral particle is a lentiviralparticle. Such retroviral particle typically includes a retroviralgenome within a capsid which is located within a viral envelope.

In some embodiments, DNA-containing viral particles are utilized insteadof recombinant retroviral particles. Such viral particles can beadenoviruses, adeno-associated viruses, herpesviruses,cytomegaloviruses, poxviruses, avipox viruses, influenza viruses,vesicular stomatitis virus (VSV), or Sindbis virus. A skilled artisanwill appreciate how to modify the methods disclosed herein for use withdifferent viruses and retroviruses, or retroviral particles. Where viralparticles are used that include a DNA genome, a skilled artisan willappreciate that functional units can be included in such genomes toinduce integration of all or a portion of the DNA genome of the viralparticle into the genome of a T cell transduced with such virus.

In some embodiments, the HIV RREs and the polynucleotide region encodingHIV Rev can be replaced with N-terminal RGG box RNA binding motifs and apolynucleotide region encoding ICP27. In some embodiments, thepolynucleotide region encoding HIV Rev can be replaced with one or morepolynucleotide regions encoding adenovirus E1B 55-kDa and E4 Orf6.

Provided herein in one aspect is a container, such as a commercialcontainer or package, or a kit comprising the same, comprising isolatedreplication incompetent recombinant retroviral particles according toany of the replication incompetent recombinant retroviral particleaspects provided herein. Furthermore, provided herein in another aspectis a container, such as a commercial container or package, or a kitcomprising the same, comprising isolated packaging cells, inillustrative embodiments isolated packaging cells from a packaging cellline, according to any of the packaging cell and/or packaging cell lineaspects provided herein. In some embodiments, the kit includesadditional containers that include additional reagents such as buffersor reagents used in methods provided herein. Furthermore provided hereinin certain aspects are use of any replication incompetent recombinantretroviral particle provided herein in any aspect, in the manufacture ofa kit for genetically modifying a T cell or NK cell according to anyaspect provided herein. Furthermore provided herein in certain aspectsare use of any packaging cell or packaging cell line provided herein inany aspect, in the manufacture of a kit for producing the replicationincompetent recombinant retroviral particles according to any aspectprovided herein.

Provided herein in one aspect is a commercial container containing areplication incompetent recombinant retroviral particle and instructionsfor the use thereof to treat tumor growth in a subject, wherein thereplication incompetent recombinant retroviral particle comprises in itsgenome a polynucleotide comprising one or more nucleic acid sequencesoperatively linked to a promoter active in T cells and/or NK cells. Insome embodiments, a nucleic acid sequence of the one or more nucleicacid sequences can encode a chimeric antigen receptor (CAR) comprisingan antigen-specific targeting region (ASTR), a transmembrane domain, andan intracellular activating domain. In some embodiments, a nucleic acidsequence of the one or more nucleic acid sequences can encode two ormore inhibitory RNA molecules directed against one or more RNA targets.

The container that contains the recombinant retroviral particles can bea tube, vial, well of a plate, or other vessel for storage of arecombinant retroviral particle. The kit can include two or morecontainers wherein a second or other container can include, for example,a solution or media for transduction of T cells and/or NK cells, and/ora the second or other container can include a pH-modulatingpharmacologic agent. Any of these containers can be of industrialstrength and grade. The replication incompetent recombinant retroviralparticle in such aspects that include a kit and a nucleic acid encodingan inhibitory RNA molecule, can be any of the embodiments for suchreplication incompetent recombinant retroviral particles providedherein, which include any of the embodiments for inhibitory RNA providedherein.

In another aspect, provided herein is the use of a replicationincompetent recombinant retroviral particle in the manufacture of a kitfor genetically modifying a T cell or NK cell, wherein the use of thekit includes: contacting the T cell or NK cell ex vivo with thereplication incompetent recombinant retroviral particle, wherein thereplication incompetent recombinant retroviral particle includes apseudotyping element on a surface and a T cell activation element on thesurface, wherein said contacting facilitates transduction of the T cellor NK cell by the replication incompetent recombinant retroviralparticle, thereby producing a genetically modified T cell or NK cell. Insome embodiments, the T cell or NK cell can be from a subject. In someembodiments, the T cell activation element can be membrane-bound. Insome embodiments, the contacting can be performed for between 1, 2, 3,4, 5, 6, 7, or 8 hours on the low end of the range and 4, 5, 6, 7, 8,10, 12, 15, 18, 21, and 24 hours on the high end of the range, forexample, between 1 and 12 hours. The replication incompetent recombinantretroviral particle for use in the manufacture of a kit can include anyof the aspects, embodiments, or subembodiments discussed elsewhereherein.

Genetically Modified T Cells and Nk Cells

In embodiments of the methods and compositions herein, geneticallymodified lymphocytes are produced, which themselves are a separateaspect of the invention. Such genetically modified lymphocytes can betransduced lymphocytes. In one aspect, provided herein is a geneticallymodified T cell or NK cell wherein the T cell or NK cell has beengenetically modified to express a first engineered signalingpolypeptide. In some embodiments, the first engineered signalingpolypeptide can be a lymphoproliferative element or a CAR that includesan antigen-specific targeting region (ASTR), a transmembrane domain, andan intracellular activating domain. In some embodiments, the T cell orNK cell can further include a second engineered signaling polypeptidethat can be a CAR or a lymphoproliferative element. In some embodiments,the lymphoproliferative element can be a chimeric lymphoproliferativeelement. In some embodiments, the T cell or NK cell can further includea pseudotyping element on a surface. In some embodiments, the T cell orNK cell can further include an activation element on a surface. The CAR,lymphoproliferative element, pseudotyping element, and activationelement of the genetically modified T cell or NK cell can include any ofthe aspects, embodiments, or subembodiments disclosed herein. Inillustrative embodiments, the activation element can be anti-CD3 scFvFc.

Some aspects provided herein include a genetically modified T cell or NKcell made by transducing resting T cells and/or resting NK cellsaccording to a method comprising contacting the resting T cells and/orresting NK cells ex vivo, with replication incompetent recombinantretroviral particles, wherein the replication incompetent recombinantretroviral particles comprise a pseudotyping element on their surfaceand a membrane-bound T cell activation element on their surface, whereinsaid contacting facilitates transduction of the resting T cells and/orNK cells by the replication incompetent recombinant retroviralparticles, thereby producing genetically modified T cells and/or NKcells, and wherein the contacting is performed for between 1, 2, 3, 4 or6 hours on the low end of the range and 4, 6, 8, 10, 12, 18, 20, or 24hours on the high end of the range, for example between 1 hour and 12hours.

In some embodiments, genetically modified lymphocytes are lymphocytessuch as T cells or NK cells that have been genetically modified toexpress a first engineered signaling polypeptide comprising at least onelymphoproliferative element and/or a second engineered signalingpolypeptide comprising a chimeric antigen receptor, which includes anantigen-specific targeting region (ASTR), a transmembrane domain, and anintracellular activating domain. In some embodiments of any of theaspects herein, the NK cells are NKT cells. NKT cells are a subset of Tcells that express CD3 and typically coexpress an αβ T-cell receptor,but also express a variety of molecular markers that are typicallyassociated with NK cells (such as NK1.1 or CD56).

Genetically modified lymphocytes of the present disclosure possess aheterologous nucleic acid sequence that has been introduced into thelymphocyte by a recombinant DNA method. For example, the heterologoussequence in illustrative embodiments is inserted into the lymphocyteduring a method for transducing the lymphocyte provided herein. Theheterologous nucleic acid is found within the lymphocyte and in someembodiments is or is not integrated into the genome of the geneticallymodified lymphocyte.

In illustrative embodiments, the heterologous nucleic acid is integratedinto the genome of the genetically modified lymphocyte. Such lymphocytesare produced, in illustrative embodiments, using a method fortransducing lymphocytes provided herein, that utilizes a recombinantretroviral particle. Such recombinant retroviral particle can include apolynucleotide that encodes a chimeric antigen receptor that typicallyincludes at least an antigen-specific targeting region (ASTR), atransmembrane domain, and an intracellular activating domain. Providedherein in other sections of this disclosure are various embodiments ofreplication incompetent recombinant retroviral particles andpolynucleotides encoded in a genome of the replication incompetentretroviral particle, that can be used to produce genetically modifiedlymphocytes that themselves form another aspect of the presentdisclosure.

Genetically modified lymphocytes of the present disclosure can beisolated outside the body. For example, such lymphocytes can be found inmedia and other solutions that are used for ex vivo transduction asprovided herein. The lymphocytes can be present in a geneticallyunmodified form in blood that is collected from a subject in methodsprovided herein, and then genetically modified during method oftransduction. The genetically modified lymphocytes can be found inside asubject after they are introduced or reintroduced into the subject afterthey have been genetically modified. The genetically modifiedlymphocytes can be a resting T cell or a resting NK cell, or thegenetically modified T cell or NK cell can be actively dividing,especially after it expresses some of the functional elements providedin nucleic acids that are inserted into the T cell or NK cell aftertransduction as disclosed herein.

Provided herein in one aspect is a transduced and/or geneticallymodified T cell or NK cell, comprising a recombinant polynucleotidecomprising one or more transcriptional units operatively linked to apromoter active in T cells and/or NK cells, in its genome, thatexpresses one or more of the functional elements provided in any of theaspects and embodiments of the present disclosure. For example, the oneor more transcriptional units can express a CAR, which can include anyof the CAR elements provided herein such as an ASTR, as a non-limitingexample a MBR-ASTR, a transmembrane domain, and an intracellularsignaling domain, and can further include as non-limiting example, amodulatory domain. Furthermore, the functional element(s) expressedwithin the transduced and/or genetically modified T cell or NK cell, oneor more of the lymphoproliferative elements provided herein, for examplea constitutively active IL-7 receptor mutant or otherlymphoproliferative element that is not an inhibitory RNA molecule (e.g.an miRNA or an shRNA), a recognition and/or elimination domain.

In one aspect, provided herein is a genetically modified T cell or NKcell comprising:

-   -   a. one or more (e.g. two or more) inhibitory RNA molecules        directed against one or more RNA targets; and    -   b. a chimeric antigen receptor (CAR) comprising an        antigen-specific targeting region (ASTR), a transmembrane        domain, and an intracellular activating domain,

and/or nucleic acids encoding the inhibitory RNA molecules directedagainst one or more RNA targets and the CAR, wherein said one (e.g. two)or more inhibitory RNA molecules and the CAR, or the nucleic acidsencoding the same are encoded by or are nucleic acid sequences that aregenetic modifications of the T cell and/or NK cell.

The genetically modified T cell or NK cell can be a population ofgenetically modified T cells and/or NK cells that include the one (e.g.two) or more inhibitory RNA molecules directed against one or more RNAtargets; and the CAR.

In some embodiments of the aspect immediately above where the T cell orNK cell comprises one or more (e.g. two or more) inhibitory RNAmolecules and the CAR, or nucleic acids encoding the same, any of thespecific embodiments provided herein for elements that can be includedas part of the CAR or that can be expressed along with alymphoproliferative element or used to control a lymphoproliferativeelement can be included.

In some embodiments of the aspect immediately above where the T cell orNK cell comprises one or more (e.g. two or more) inhibitory RNAmolecules and the CAR, or nucleic acids encoding the same, the CAR is amicroenvironment restricted biologic (MRB)-CAR and/or the geneticallymodified T cell or NK cell can further include at least onelymphoproliferative element that is not an inhibitory RNA molecule,and/or a nucleic acid encoding the lymphoproliferative element. In suchembodiments, the lymphoproliferative element is encoded by nucleic acidsequences that are genetic modifications of the T cell and/or NK cell.Any of the lymphoproliferative elements disclosed herein can be usedand/or encoded for, in such embodiments. For example, the at least onelymphoproliferative element can be a constitutively active IL-7receptor.

In some embodiments of the aspect immediately above where the T cell orNK cell comprises one or more (e.g. two or more) inhibitory RNAmolecules and the CAR, or nucleic acids encoding the same, theinhibitory RNA molecule is a precursor of a miRNA or an shRNA. In someembodiments of this aspect the one (e.g. two) or more inhibitory RNAmolecules are polycistronic. In some embodiments of this aspect the one(e.g. two) or more inhibitory RNA molecules are directed against thesame or in illustrative embodiments, different RNA targets. In someembodiments of this aspect, one, most or all of the one (e.g. two) ormore inhibitory RNA molecules decreases expression of an endogenous TCR.

In some embodiments of the aspect immediately above where the T cell orNK cell comprises one or more (e.g. two or more) inhibitory RNAmolecules and the CAR, or nucleic acids encoding the same, the RNAtarget is mRNA transcribed from a gene selected from the groupconsisting of: PD-1, CTLA4, TCR alpha, TCR beta, CD3 zeta, SOCS, SMAD2,a miR-155 target, IFN gamma, cCBL, TRAIL2, PP2A, and ABCG1. In 144. Thegenetically modified T cell and/or NK cell of any one of claims 135-140,wherein said RNA target is mRNA transcribed from the TCR alpha gene. Insome embodiments of this aspect at least one of the one (e.g. two) ormore inhibitory RNA molecules is miR-155.

In some embodiments of the aspect immediately above where the T cell orNK cell comprises one or more (e.g. two or more) inhibitory RNAmolecules and the CAR, or nucleic acids encoding the same, the ASTR ofthe CAR is an MRB ASTR and/or the ASTR of the CAR binds to a tumorassociated antigen. Furthermore, in some embodiments of the aboveaspect, the first nucleic acid sequence is operably linked to ariboswitch, which for example is capable of binding a nucleoside analog,and in illustrative embodiments is an antiviral drug such as acyclovir.

In the methods and compositions disclosed herein, expression ofengineered signaling polypeptides is regulated by a control element, andin some embodiments, the control element is a polynucleotide comprisinga riboswitch. In certain embodiments, the riboswitch is capable ofbinding a nucleoside analog and when the nucleoside analog is present,one or both of the engineered signaling polypeptides are expressed.

The genetically modified lymphocytes disclosed herein can also havepolypeptides on their surface that are remnants of fusion of areplication incompetent recombinant retroviral particle during atransduction method provided herein. Such polypeptides can include, anactivation element, a pseudotyping element, and/or one or more fusionpolypeptides that include a cytokine.

Provided herein in one aspect, is a genetically modified T cell and/orNK cell that expresses one or more (e.g. two or more) inhibitory RNAmolecules directed against one or more RNA targets and a chimericantigen receptor, or CAR, as disclosed herein. In some embodiments, thegenetically modified T cell and/or NK cell further expresses at leastone lymphoproliferative element as disclosed herein that is not aninhibitory RNA molecule. In certain embodiments, the geneticallymodified T cell and/or NK cell also expresses one or more riboswitchesthat control expression of the one or more inhibitory RNA molecules, theCAR, and/or the at least one lymphoproliferative element that is not aninhibitory RNA molecule. In some embodiments, the genetically modified Tcell and/or NK cell expresses two to 10 inhibitory RNA molecules. Infurther embodiments, the genetically modified T cell and/or NK cellexpresses two to six inhibitory RNA molecules. In illustrativeembodiments, the genetically modified T cell and/or NK cell expressesfour inhibitory RNA molecules.

Nucleic Acids

The present disclosure provides nucleic acid encoding polypeptides ofthe present disclosure. A nucleic acid will in some embodiments be DNA,including, e.g., a recombinant expression vector. A nucleic acid will insome embodiments be RNA, e.g., in vitro synthesized RNA.

In some cases, a nucleic acid provides for production of a polypeptideof the present disclosure, e.g., in a mammalian cell. In other cases, asubject nucleic acid provides for amplification of the nucleic acidencoding a polypeptide of the present disclosure.

A nucleotide sequence encoding a polypeptide of the present disclosurecan be operably linked to a transcriptional control element, e.g., apromoter, and enhancer, etc.

Suitable promoter and enhancer elements are known in the art. Forexpression in a bacterial cell, suitable promoters include, but are notlimited to, lacl, lacZ, T3, T7, gpt, lambda P and trc. For expression ina eukaryotic cell, suitable promoters include, but are not limited to,light and/or heavy chain immunoglobulin gene promoter and enhancerelements; cytomegalovirus immediate early promoter; herpes simplex virusthymidine kinase promoter; early and late SV40 promoters; promoterpresent in long terminal repeats from a retrovirus; mousemetallothionein-I promoter; and various art-known tissue specificpromoters.

Suitable reversible promoters, including reversible inducible promotersare known in the art. Such reversible promoters may be isolated andderived from many organisms, e.g., eukaryotes and prokaryotes.Modification of reversible promoters derived from a first organism foruse in a second organism, e.g., a first prokaryote and a second aeukaryote, a first eukaryote and a second a prokaryote, etc., is wellknown in the art. Such reversible promoters, and systems based on suchreversible promoters but also comprising additional control proteins,include, but are not limited to, alcohol regulated promoters (e.g.,alcohol dehydrogenase I (alcA) gene promoter, promoters responsive toalcohol transactivator proteins (AlcR), etc.), tetracycline regulatedpromoters, (e.g., promoter systems including TetActivators, TetON,TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoidreceptor promoter systems, human estrogen receptor promoter systems,retinoid promoter systems, thyroid promoter systems, ecdysone promotersystems, mifepristone promoter systems, etc.), metal regulated promoters(e.g., metallothionein promoter systems, etc.), pathogenesis-relatedregulated promoters (e.g., salicylic acid regulated promoters, ethyleneregulated promoters, benzothiadiazole regulated promoters, etc.),temperature regulated promoters (e.g., heat shock inducible promoters(e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), lightregulated promoters, synthetic inducible promoters, and the like.

In some instances, the locus or construct or trans gene containing thesuitable promoter is irreversibly switched through the induction of aninducible system. Suitable systems for induction of an irreversibleswitch are well known in the art, e.g., induction of an irreversibleswitch may make use of a Cre-lox-mediated recombination (see, e.g.,Fuhrmann-Benzakein, et al., PNAS (2000) 28:e99, the disclosure of whichis incorporated herein by reference). Any suitable combination ofrecombinase, endonuclease, ligase, recombination sites, etc. known tothe art may be used in generating an irreversibly switchable promoter.Methods, mechanisms, and requirements for performing site-specificrecombination, described elsewhere herein, find use in generatingirreversibly switched promoters and are well known in the art, see,e.g., Grindley et al. (2006) Annual Review of Biochemistry, 567-605 andTropp (2012) Molecular Biology (Jones & Bartlett Publishers, Sudbury,MA), the disclosures of which are incorporated herein by reference.

In some cases, the promoter is a CD8 cell-specific promoter, a CD4cell-specific promoter, a neutrophil-specific promoter, or anNK-specific promoter. For example, a CD4 gene promoter can be used; see,e.g., Salmon et al. (1993) Proc. Natl. Acad. Sci. USA 90:7739; andMarodon et al. (2003) Blood 101:3416. As another example, a CD8 genepromoter can be used. NK cell-specific expression can be achieved by useof an Neri (p46) promoter; see, e.g., Eckelhart et al. (2011) Blood117:1565.

In some embodiments, e.g., for expression in a yeast cell, a suitablepromoter is a constitutive promoter such as an ADH1 promoter, a PGK1promoter, an ENO promoter, a PYK1 promoter and the like; or aregulatable promoter such as a GALI promoter, a GALlO promoter, an ADH2promoter, a PH05 promoter, a CUPl promoter, a GAL7 promoter, a MET25promoter, a MET3 promoter, a CYCl promoter, a HIS3 promoter, an ADHlpromoter, a PGK promoter, a GAPDH promoter, an ADCl promoter, a TRPlpromoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TPlpromoter, and AOXl (e.g., for use in Pichia). Selection of theappropriate vector and promoter is well within the level of ordinaryskill in the art.

Suitable promoters for use in prokaryotic host cells include, but arenot limited to, a bacteriophage T7 RNA polymerase promoter; a trppromoter; a lac operon promoter; a hybrid promoter, e.g., a lac/tachybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lacpromoter; a trc promoter; a tac promoter, and the like; an araBADpromoter; in vivo regulated promoters, such as an ssaG promoter or arelated promoter (see, e.g., U.S. Patent Publication No. 20040131637), apagC promoter (Pulkkinen and Miller, J. Bacterial., 1991: 173(1): 86-93;Alpuche-Aranda et al., PNAS, 1992; 89(21): 10079-83), a nirB promoter(Harborne et al. (1992) Mal. Micro. 6:2805-2813), and the like (see,e.g., Dunstan et al. (1999) Infect. Immun. 67:5133-5141; McKelvie et al.(2004) Vaccine 22:3243-3255; and Chatfield et al. (1992) Biotechnol.10:888-892); a sigma70 promoter, e.g., a consensus sigma70 promoter(see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); astationary phase promoter, e.g., a dps promoter, an spv promoter, andthe like; a promoter derived from the pathogenicity island SPI-2 (see,e.g., WO96/17951); an actA promoter (see, e.g., Shetron-Rama et al.(2002) Infect. Immun. 70:1087-1096); an rpsM promoter (see, e.g.,Valdivia and Falkow (1996). Mal. Microbial. 22:367); a tet promoter(see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. andHeinemann, U. (eds), Topics in Molecular and Structural Biology,Protein-Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp.143-162); an SP6 promoter (see, e.g., Melton et al. (1984) Nucl. AcidsRes. 12:7035); and the like. Suitable strong promoters for use inprokaryotes such as Escherichia coli include, but are not limited toTrc, Tac, T5, T7, and PLambda. Non-limiting examples of operators foruse in bacterial host cells include a lactose promoter operator (Lacirepressor protein changes conformation when contacted with lactose,thereby preventing the Laci repressor protein from binding to theoperator), a tryptophan promoter operator (when complexed withtryptophan, TrpR repressor protein has a conformation that binds theoperator; in the absence of tryptophan, the TrpR repressor protein has aconformation that does not bind to the operator), and a tac promoteroperator (see, for example, deBoer et al. (1983) Proc. Natl. Acad. Sci.U.S.A. 80:21-25).

A nucleotide sequence encoding a polypeptide of the disclosure can bepresent in an expression vector and/or a cloning vector. Nucleotidesequences encoding two separate polypeptides can be cloned in the sameor separate vectors. An expression vector can include a selectablemarker, an origin of replication, and other features that provide forreplication and/or maintenance of the vector. Suitable expressionvectors include, e.g., plasmids, viral vectors, and the like.

Large numbers of suitable vectors and promoters are known to those ofskill in the art; many are commercially available for generating asubject recombinant constructs. The following bacterial vectors areprovided by way of example: pBs, phagescript, PsiX174, pBluescript SK,pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, CA, USA);pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala,Sweden). The following eukaryotic vectors are provided by way ofexample: pWLneo, pSV2cat, pOG44, PXRl, pSG (Stratagene) pSVK3, pBPV,pMSG, and pSVL (Pharmacia).

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host may be present.

As noted above, in some embodiments, a nucleic acid encoding apolypeptide of the present disclosure will in some embodiments be RNA,e.g., in vitro synthesized RNA. Methods for in vitro synthesis of RNAare known in the art; any known method can be used to synthesize RNAincluding a nucleotide sequence encoding a polypeptide of the presentdisclosure. Methods for introducing RNA into a host cell are known inthe art. See, e.g., Zhao et al. (2010) Cancer Res. 15:9053. IntroducingRNA including a nucleotide sequence encoding a polypeptide of thepresent disclosure into a host cell can be carried out in vitro or exvivo or in vivo. For example, a host cell (e.g., an NK cell, a cytotoxicT lymphocyte, etc.) can be electroporated in vitro or ex vivo with RNAcomprising a nucleotide sequence encoding a polypeptide of the presentdisclosure.

Various aspects and embodiments that include a polynucleotide, a nucleicacid sequence, and/or a transcriptional unit, and/or a vector includingthe same, further include one or more of a Kozak-type sequence, awoodchuck hepatitis virus post-transcriptional regulatory element(WPRE), and a double stop codon or a triple stop codon, wherein one ormore stop codons of the double stop codon or the triple stop codondefine a termination of a reading from of at least one of the one ormore transcriptional units. In certain embodiments, a polynucleotide anucleic acid sequence, and/or a transcriptional unit, and/or a vectorincluding the same, further includes a Kozak-type sequence having a 5′nucleotide within 10 nucleotides upstream of a start codon of at leastone of the one or more transcriptional units. In one embodiment theKozak-type sequence is or includes CCACCAT/UG(G) (SEQ ID NO:515),CCGCCAT/UG(G) (SEQ ID NO:516), GCCGCCGCCAT/UG(G) (SEQ ID NO:517), orGCCGCCACCAT/UG(G) (SEQ ID NO:515) (with nucleotides in parenthesisrepresenting optional nucleotides and nucleotides separated by a slashindicated different possible nucleotides at that position, for exampledepending on whether the nucleic acid is DNA or RNA. In theseembodiments that include the AU/TG start codon, the A can be consideredposition 0. In certain illustrative embodiments, the nucleotides at −3and +4 are identical, for example the −3 and +4 nucleotides can be G. Inanother embodiment the Kozak-type sequence includes an A or G in the 3rdposition upstream of ATG where ATG is the start codon. In anotherembodiment the Kozak-type sequence includes an A or G in the 3rdposition upstream of AUG where AUG is the start codon. In anillustrative embodiment, the Kozak sequence is (GCC)GCCRCCATG, where Ris a purine (A or G). In an illustrative embodiment, the Kozak-typesequence is GCCGCCACCAUG (SEQ ID NO:518). In another embodiment, whichcan be combined with the preceding embodiment that includes a Kozak-typesequence and/or the following embodiment that includes triple stopcodon, the polynucleotide includes a WPRE element. WPREs have beencharacterized in the art (See e.g., (Higashimoto et al., Gene Ther.2007; 14: 1298)) and exemplified in Examples 17 and 18 herein. In someembodiments, the WPRE element is located 3′ of a stop codon of the oneor more transcriptional units and 5′ to a 3′ LTR of the polynucleotide.In another embodiment, which can be combined with either or both of thepreceding embodiments (i.e. an embodiment the polynucleotide includes aKozak-type sequence and/or an embodiment wherein the polynucleotideincludes a WPRE), the one or more transcriptional units terminates withone or more stop codons of a double stop codon or a triple stop codon,wherein the double stop codon includes a first stop codon in a firstreading frame and a second stop codon in a second reading frame, or afirst stop codon in frame with a second stop codon, and wherein thetriple stop codon includes a first stop codon in a first reading frame,a second stop codon in a second reading frame, and a third stop codon ina third reading frame, or a first stop codon in frame with a second stopcodon and a third stop codon.

A triple stop codon herein includes three stop codons, one in eachreading frame, within 10 nucleotides of each other, and preferablyhaving overlapping sequence, or three stop codons in the same readingframe, preferably at consecutive codons. A double stop codon means twostop codons, each in a different reading frame, within 10 nucleotides ofeach other, and preferably having overlapping sequences, or two stopcodons in the same reading frame, preferably at consecutive codons.

In some of the methods and compositions disclosed herein, theintroduction of DNA into PBMCs, B cells, T cells and/or NK cells andoptionally the incorporation of the DNA into the host cell genome, isperformed using methods that do not utilize replication incompetentrecombinant retroviral particles. For example, other viral vectors canbe utilized, such as those derived from adenovirus, adeno-associatedvirus, or herpes simplex virus-1, as non-limiting examples.

In some embodiments, methods provided herein can include transfectingand/or transducing target cells with non-viral vectors. In any of theembodiments disclosed herein that utilize non-viral vectors to transfecttarget cells, the non-viral vectors, including naked DNA, can beintroduced into the target cells, such as for example, PBMCs, B cells, Tcells and/or NK cells using methods that include electroporation,nucleofection, liposomal formulations, lipids, dendrimers, cationicpolymers such as poly(ethylenimine) (PEI) and poly(l-lysine) (PLL),nanoparticles, cell-penetrating peptides, microinjection, and/ornon-integrating lentiviral vectors. In some embodiments, DNA can beintroduced into target cells, such as PBMCs, B cells, T cells and/or NKcells in a complex with liposomes and protamine. Other methods fortransfecting and/or transducing T cells and/or NK cells ex vivo that canbe used in embodiments of methods provided herein, are known in the art(see e.g., Morgan and Boyerinas, Biomedicines. 2016 Apr. 20; 4(2). pii:E9, incorporated by reference herein in its entirety).

In some embodiments of method provided herein, DNA can be integratedinto the genome using transposon-based carrier systems byco-transfection, co-nucleofection or co-electroporation of target DNA asplasmid containing the transposon ITR fragments in 5′ and 3′ ends of thegene of interest and transposase carrier system as DNA or mRNA orprotein or site specific serine recombinases such as phiC31 thatintegrates the gene of interest in pseudo attP sites in the humangenome, in this instance the DNA vector contains a 34 to 40 bp attB sitethat is the recognition sequence for the recombinase enzyme (BhaskarThyagarajan et al. Site-Specific Genomic Integration in Mammalian CellsMediated by Phage φC31 Integrase, Mol Cell Biol. 2001 June; 21(12):3926-3934) and co transfected with the recombinase. For T cells and/orNK cells, transposon-based systems that can be used in certain methodsprovided herein utilize the Sleeping Beauty DNA carrier system (seee.g., U.S. Pat. No. 6,489,458 and U.S. patent application Ser. No.15/434,595, incorporated by reference herein in their entireties), thePiggyBac DNA carrier system (see e.g., Manuri et al., Hum Gene Ther.2010 April; 21(4):427-37, incorporated by reference herein in itsentirety), or the Tol2 transposon system (see e.g., Tsukahara et al.,Gene Ther. 2015 February; 22(2): 209-215, incorporated by referenceherein in its entirety) in DNA, mRNA, or protein form. In someembodiments, the transposon and/or transposase of the transposon-basedvector systems can be produced as a minicircle DNA vector beforeintroduction into T cells and/or NK cells (see e.g., Hudecek et al.,Recent Results Cancer Res. 2016; 209:37-50 and Monjezi et al., Leukemia.2017 Jan.; 31(1):186-194, incorporated by reference herein in theirentireties). The CAR or lymphoproliferative element can also beintegrated into the defined and specific sites in the genome usingCRISPR or TALEN mediated integration, by adding 50-1000 bp homology armshomologous to the integration 5′ and 3′ of the target site (Jae SeongLee et al. Scientific Reports 5, Article number: 8572 (2015),Site-specific integration in CHO cells mediated by CRISPR/Cas9 andhomology-directed DNA repair pathway). CRISPR or TALEN providespecificity and genomic-targeted cleavage and the construct will beintegrated via homology-mediated end joining (Yao X at al. Cell Res.2017 Jun.; 27(6):801-814. doi: 10.1038/cr.2017.76. Epub 2017 May 19).The CRISPR or TALEN can be co-transfected with target plasmid as DNA,mRNA, or protein.

Packaging Cells

The present disclosure provides packaging cells and mammalian cell linesthat are packaging cell lines that produce replication incompetentrecombinant retroviral particles that genetically modify targetmammalian cells and the target mammalian cells themselves. Inillustrative embodiments, the packaging cell comprises nucleic acidsequences encoding a packagable RNA genome of the replicationincompetent retroviral particle, a REV protein, a gag polypeptide, a polpolypeptide, and a pseudotyping element.

Suitable mammalian cells include primary cells and immortalized celllines. Suitable mammalian cell lines include human cell lines, non-humanprimate cell lines, rodent (e.g., mouse, rat) cell lines, and the like.Suitable mammalian cell lines include, but are not limited to, HeLacells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHOcells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), HEK293 cells (e.g.,ATCC No. CRL-1573), suspension-adapted HEK293 cells, Vero cells, NIH 3T3cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK

cells (e.g., ATCC No. CCLlO), PC12 cells (ATCC No. CRL1721), COS cells,COS-7 cells (ATCC No. CRL1651), RATl cells, mouse L cells (ATCC No.CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS),and the like.

In some instances, the cell is not an immortalized cell line, but isinstead a cell (e.g., a cell) obtained from an individual or an ex vivocell. For example, in some cases, the cell is an immune cell obtainedfrom an individual. As another example, the cell is a stem cell orprogenitor cell obtained from an individual. In any of the embodimentsand aspects provided herein, that include a B cell, T cell, or NK cell,the cell can be an autologous cell or it can be an allogeneic cell (alsocalled allogenic cell herein). In some embodiments the allogenic cellcan be a genetically engineered allogenic cell. Allogeneic cells, suchas allogenic T cells, and methods for genetically engineering allogeneiccells, are known in the art. In some embodiments, the allogeneic cellcan be an immortalized cell. In some embodiments where the allogeneiccell is a T cell, the T cell has been genetically engineered such thatat least one of its T cell receptor chains does not function and/or isat least partially deleted. In some embodiments the allogeneic cell canbe modified such that it is missing all or part of the B2 microglobulingene. In some embodiments where allogeneic cells are used in methodsherein, lymphoproliferative elements and/or CLEs can function to reducethe number of cells that are necessary for practicing an inventionherein and can facilitate cell manufacturing of T cells, B cells, or NKcells from a subject.

Methods of Activating an Immune Cell

The present disclosure provides methods of activating an immune cell, inillustrative embodiments a lymphocyte, in vitro, in vivo, or ex vivo.The methods generally involve contacting an immune cell (in vitro, invivo, or ex vivo) with an activating element, or with one or more targetantigens, where the immune cell has been genetically modified to producea microenvironment restricted CAR of the present disclosure. In thepresence of the one or more target antigens, the microenvironmentrestricted CAR activates the immune cell, thereby producing an activatedimmune cell. Immune cells include, e.g., a cytotoxic T lymphocyte, an NKcell, a CD4⁺ T cell, a T regulatory (Treg) cell, a γδ T cell, an NK-Tcell, neutrophils, etc. In other embodiments, contacting a T cell with aT cell activating agent, activates the T cell. Such methods are providedherein and discussed in the Activation Element section herein.

Contacting the genetically modified immune cell (e.g., a T lymphocyte,an NK cell) with one or more target antigens can increase production ofa cytokine by the immune cell by at least about 10%, at least about 15%,at least about 20%, at least about 25%, at least about 30%, at leastabout 40%, at least about 50%, at least about 75%, at least about2-fold, at least about 2.5-fold, at least about 5-fold, at least about10-fold, or more than 10-fold, compared with the amount of cytokineproduced by the immune cell in the absence of the one or more targetantigens. Cytokines whose production can be increased include, but arenot limited to, IL-2 and IFN-γ.

Contacting a genetically modified cytotoxic cell (e.g., cytotoxic Tlymphocyte) with AAR can increase cytotoxic activity of the cytotoxiccell by at least about 10%, at least about 15%, at least about 20%, atleast about 25%, at least about 30%, at least about 40%, at least about50%, at least about 75%, at least about 2-fold, at least about 2.5-fold,at least about 5-fold, at least about 10-fold, or more than 10-fold,compared to the cytotoxic activity of the cytotoxic cell in the absenceof the one or more target antigens.

Contacting a genetically modified cytotoxic cell (e.g., cytotoxic Tlymphocyte) with one or more target antigens can increase cytotoxicactivity of the cytotoxic cell by at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 40%, at least about 50%, at least about 75%, at least about2-fold, at least about 2.5-fold, at least about 5-fold, at least about10-fold, or more than 10-fold, compared to the cytotoxic activity of thecytotoxic cell in the absence of the one or more target antigens.

In other embodiments, e.g., depending on the host immune cell,contacting a genetically modified host cell with an antigen can increaseor decrease cell proliferation, cell survival, cell death, and the like.

Methods for Making a Microenvironment Restricted Antigen-SpecificTargeting Region

In some embodiments, antigen binding domains (also referred to herein as“antigen-specific target regions” or “ASTRs”) of CARs constitutivelybind their cognate antigens. In other embodiments, the ASTRs can bemicroenvironment restricted, preferentially or only binding theircognate antigen under certain aberrant conditions, such as those thatexist in the tumor microenvironment, as disclosed in more detail herein.Microenvironment restricted ASTRs that bind preferentially orexclusively under aberrant conditions of a tumor microenvironment, canprovide a reduction in on-target off-tumor effects as binding to theantigen in normal physiological conditions is reduced, in somesituations to levels below detection by immunoassays. In certainaspects, CARs provided herein include a microenvironment restricted ASTRthat specifically binds to a target protein, wherein the ASTR is an scFvfragment that includes a heavy chain variable region and a light chainvariable region.

Certain illustrative embodiments of the aspects disclosed herein, forexample the methods, cells, cells lines, replication incompetentrecombinant retroviral particles, polynucleotides, or vectors disclosedherein, include CARs that include microenvironment restrictedantigen-specific targeting regions.

Accordingly, in one aspect, provided herein is a chimeric antigenreceptor for binding a target antigen, that includes:

-   -   a) a microenvironment restricted antigen-specific targeting        region that exhibits an increase in binding to the target        antigen in an aberrant condition compared to a normal        physiological environment, wherein the antigen-specific        targeting region binds to the target;    -   b) a transmembrane domain; and    -   c) an intracellular activating domain.

In another aspect, provided herein is a chimeric antigen receptor forbinding a target antigen, that includes:

-   -   a) at least one microenvironment restricted antigen specific        targeting region selected by panning a polypeptide library and        having an increase in activity in a target antigen binding assay        at an aberrant condition compared to a normal physiological        condition;    -   b) a transmembrane domain; and    -   c) an intracellular activating domain.

In some embodiments of any aspect disclosed herein, any of the chimericantigen receptors can be microenvironment restricted such that theyexhibit an increase in binding activity at an aberrant conditioncompared to a normal physiological condition. In some illustrativeembodiments of any aspect disclosed herein, the microenvironmentrestricted ASTR is identified from an initial polypeptide librarywithout mutating/evolving members of the library beforescreening/evolving and/or without mutating during or between optionalrepeated rounds of screening. Exemplary transmembrane domains andintracellular activating domains can be any of those disclosed hereinfor CARs.

In one aspect, provided herein is a method for selecting amicroenvironment restricted ASTR, comprising panning a polypeptidedisplay library by:

-   -   a. subjecting polypeptides of the polypeptide display library to        a target antigen binding assay under a normal physiological        condition and a target antigen binding assay under an aberrant        condition; and    -   b. selecting a polypeptide which exhibits an increase in target        antigen binding activity at the aberrant condition compared to        the physiological condition, thereby selecting the        microenvironment restricted antigen specific targeting region.

In another aspect, provided herein is a method for isolating amicroenvironment restricted ASTR, that includes panning a polypeptidelibrary by:

-   -   contacting the polypeptide library under aberrant conditions        with a target antigen bound to a solid support, wherein clones        expressing polypeptides that bind the target antigen remain        bound to the solid support through the target antigen;    -   incubating the solid supports with bound polypeptides under        physiological conditions; and collecting clones that elute from        the solid support under the physiological conditions, thereby        isolating the microenvironment restricted antigen-specific        targeting region.

In some illustrative embodiments of any aspect disclosed herein, themicroenvironment restricted antigen-specific targeting region isidentified from an initial polypeptide library screen withoutmutating/evolving members of the library before screening and/or withoutmutating/evolving during or between optional repeated rounds ofscreening or panning.

Normal physiological conditions can include those of temperature, pH,osmotic pressure, osmolality, oxidative stress, and electrolyteconcentration that would be considered within a normal range at the siteof administration, or at the tissue or organ at the site of action, to asubject. An aberrant condition is that which deviates from the normallyacceptable range for that condition. In one aspect, a microenvironmentrestricted antigen-specific targeting region (i.e. polypeptide) isvirtually inactive at normal conditions but is active at other thannormal conditions at a level that is equal or better than at normalconditions. For example, in one aspect, the microenvironment restrictedantigen-specific targeting region is virtually inactive at bodytemperature, but is active at lower temperatures. In another aspect, themicroenvironment restricted antigen-specific targeting region isreversibly or irreversibly inactivated at the normal conditions. In afurther aspect, the microenvironment restricted antigen-specifictargeting region is a therapeutic protein. In another aspect, themicroenvironment restricted antigen-specific targeting region is used asa drug, or therapeutic agent. In yet another aspect, themicroenvironment restricted antigen-specific targeting region is more orless active in highly oxygenated blood, such as, for example, afterpassage through the lung or in the lower pH environments found in thekidney.

In some embodiments, a single round of selection is performed to obtainthe microenvironment restricted antigen-specific targeting region. Incertain embodiments, the screening or panning method is repeated afteridentifying free polypeptides that bound antigen under aberrantconditions and did not bind under physiological conditions, or cellsexpressing a test polypeptide that had these properties, or phage coatedwith a test polypeptide that has such properties in an initial orprevious round. In some methods, phage that are collected are used toinfect cells, which can be infected with helper phage as well, in orderto amplify the collected phage. In other methods where polypeptides onthe surface of cells are tested, collected cells can be grown to“amplify” the polypeptides expressed by the cells by amplifyingpolynucleotides in the cells that encode the polypeptides. In someembodiments, the amplifying is done by growing cells that express theidentified polypeptides without performing a process to mutate thepolynucleotides encoding the identified polypeptides between rounds.Thus, polypeptides that were collected in a previous round are enrichedby amplifying cells that contain polynucleotides encoding thesecollected polypeptides.

The panning or screening method can be performed a single time, orrepeated for 1 to 1000 times. In illustrative embodiments, the panningis repeated 1 to 20 times or 2 to 10 times or 2 to 5 times.

In other methods, microenvironment restricted ASTRs against an antigenof interest (i.e. target antigen) are performed using one or more roundsof mutation/evolution between rounds of panning. In one method, awild-type protein is identified for example by generating a polypeptideor protein library and screening the polypeptide or protein library fora polypeptide or protein with a desired binding affinity to a targetantigen. In some embodiments where the wild-type proteins areantibodies, the wild-type antibodies can be discovered by generating andscreening polyclonal or monoclonal antibody libraries, including phagedisplay antibody libraries, for example phage display humanized antibodylibraries.

Evolved ASTRs can be generated by subjecting the wild-type protein, or anucleic acid sequence encoding the wild-type protein, to a process ofmutagenesis to produce a population of mutant polypeptides that can bescreened to identify a mutant ASTR with an increased activity (e.g.enhanced binding affinity to the target antigen) in a tumor environmentand/or in an in vitro tumor surrogate assay condition, compared to anormal physiological environment. Examples of such methods are providedin WO2016033331 (“CONDITIONALLY ACTIVE CHIMERIC ANTIGEN RECEPTORS FORMODIFIED T CELLS”) or U.S. Pat. No. 8,709,755, both herein incorporatedby reference in their entirety. This method of generating amicroenvironment restricted antibody is hereby incorporated by referencein its entirety herein.

In other embodiments, microenvironment restricted antigen-specificpolypeptides (i.e. targeting regions, e.g. antibodies) can be identifiedby screening an initial polypeptide library under aberrant versusphysiological conditions and identifying a test polypeptide from theinitial polypeptide library, that binds preferentially or exclusivelyunder aberrant vs. physiological conditions. In some examples, theidentified and isolated microenvironment restricted antigen-specificpolypeptides (i.e. targeting regions, e.g. antibodies) identified froman initial polypeptide library in an initial polypeptide library screen,bind their cognate antigen preferentially or exclusively under aberrantvs. physiological conditions. In such instances, no rounds ofmutating/evolving are performed. Accordingly, the method in illustrativeembodiments is performed without mutating polynucleotides encoding theisolated microenvironment restricted antigen-specific targeting regionbetween rounds of screening (e.g. rounds of panning), or performed foronly a single binding assay under aberrant versus physiologicalconditions to isolate and identify the microenvironment restrictedantigen-specific polypeptide (i.e. targeting region, e.g. antibody). Themethod can be performed by culturing, high fidelity amplifying, and/ordiluting polynucleotides encoding antigen-specific targeting regions, orhost organisms including the same, between rounds of screening and/orpanning, without any mutating/evolving. Furthermore, the method can beperformed without repeating the screening and/or panning and can beperformed without mutating/evolving a polynucleotide encoding theisolated microenvironment restricted antigen-specific targeting region,after the microenvironment restricted antigen-specific polypeptide (i.e.target region, e.g. antibody) is isolated.

Assays for use in the methods provided herein to detect binding of apolypeptide to a cognate binding partner include cell based assays, andin particular assays performed using cell surface display systems, suchas mammalian cell surface display systems. In an exemplary method,nucleic acids encoding a polypeptide or a library of variantpolypeptides, including a library of modified polypeptides, can beintroduced into a vector suitable for expression in cells, such asmammalian cells. Cells are then transfected with the vector, and thepolypeptide(s) is/are expressed by the cells. The library of cellscontaining surface-expressed polypeptides can be contacted with asolution containing a soluble or surface-bound cognate binding partner.Binding activity can be detected using any assay that can detect thebinding to the surface of the cells. Activity also can be assessed byassessing a functional activity of the polypeptide or polypeptide. Anycell based assay known to the skilled artisan is contemplated for use inthe methods provided herein, including cell proliferation assays, celldeath assays, flow cytometry, cell separation techniques, fluorescenceactivated cell sorting (FACS), phase microscopy, fluorescencemicroscopy, receptor binding assays, cell signaling assays,immunocytochemistry and reporter gene assays. In some examples, theassays are fluorescence activated cell sorting (FACS) assays.

Polypeptides or proteins can be expressed by mammalian cells assecreted, soluble molecules, cell surface molecules, or intracellularantibodies. In an exemplary method, cells can be transfected with alibrary of proteins under conditions whereby most or all of the cellsdisplay a member of the protein library anchored on the cell surface.Optionally, an expression system can be used in which most of mammaliancell transfectants have only one plasmid integrated in their genome.Therefore, most (i.e., at least about 70% or about 80% or about 90%) ofthe transfectants express one or more molecules of one polypeptide. Thiscan be verified, for example, by isolating and culturing individualtransfectants; and amplifying and sequencing the expressed sequences todetermine whether they have a single sequence.

In some examples of the methods provided herein, the polypeptides areantibodies displayed on the surface of mammalian cells. Any antibodydescribed herein can be expressed on the surface of mammalian cells,including full length, bivalent, functional antibodies, such as IgGantibodies. The antibody can be a fragment, for example, Fab fragmentsor scFv fragments. Antibodies can include an Fc region, such as anscFv-Fc or a full length antibody, which comprises two heavy and twolight chains. The skilled artisan can select a suitable antibodyfragment. For example, an ScFv-Fcs and full length antibodies made inmammalian cells can have several advantages over scFv's or Fabfragments.

Solid supports that can be used in the binding assays provided hereininclude any carrier that is capable of being affixed with a bindingpartner of a polypeptide such as a ligand, receptor or antigen.Typically, to facilitate high throughput screening a cognate bindingpartner is affixed to the solid support. Examples of carriers for use assolid supports in the methods provided herein include, but are notlimited to, glass, polystyrene, polypropylene, polyethylene, dextran,nylon, amyloses, natural and modified celluloses, polyacrylamides,agaroses and magnetic solid supports, such as solid supports thatinclude magnetite. The solid support can be one or more beads orparticles, microspheres, a surface of a tube or plate, a filtermembrane, and other solid supports known in the art. Exemplary solidsupport systems include, but are not limited to, a flat surfaceconstructed, for example, of glass, silicon, metal, nylon, cellulose,plastic or a composite, including multiwell plates or membranes; or canbe in the form of a bead such as a silica gel, a controlled pore glass,a magnetic or cellulose bead. Further, such methods can be adapted foruse in suspension or in the form of a column. In some embodiments, themicroenvironment restricted antigen-specific polypeptide (i.e. targetregion, e.g. antibody) is identified and isolated by biopanning a phagedisplay or yeast surface display (Colby et al., “Engineering AntibodyAffinity by Yeast Surface Display,” Meth. Enzym. 388, 26 (2004))antibody (e.g. humanized antibody) library with an immobilized targetantigen. For example, either a naïve humanized antibody library or asynthetic humanized antibody library can be panned using the phagedisplay or yeast surface display methods herein. In some embodiments, aninitial phage display process, phage clones can be transferred to amammalian vector and used to a mammalian cell surface screening method(See e.g., Yoon et al., BMC Biotechnology 12:62; 1472-6750 (2012)). Anexemplary method for performing phage display to isolate amicroenvironment restricted antigen-specific target region is providedin Example 2.

A microenvironment restricted ASTR identified using methods providedherein, can be an antibody, an antigen, a ligand, a receptor bindingdomain of a ligand, a receptor, a ligand binding domain of a receptor,or an affibody. In embodiments where the microenvironment restrictedASTR is an antibody, it can be a full-length antibody, a single-chainantibody, an Fab fragment, an Fab′ fragment, an (Fab′)2 fragment, an Fvfragment, and a divalent single-chain antibody or a diabody, wherein theantigen-specific targeting region comprises a heavy chain and a lightchain from an antibody. In some embodiments, the microenvironmentrestricted ASTR is a single-chain variable fragment. Such single-chainvariable fragment can have heavy and light chains separated by a linker,wherein the linker is between 6 and 100 amino acids in length. In someembodiments the heavy chain is positioned N-terminal to the light chainon the chimeric antigen receptor. In other embodiments, the light chainis positioned N-terminal to the heavy chain. The microenvironmentrestricted ASTR can be a bispecific ASTR.

Microenvironment restricted ASTRs identified using methods providedherein are typically polypeptides and more specifically polypeptideantibodies, and in illustrative embodiments, single chain antibodies.These polypeptides can bind to their cognate antigens with higher orlower affinity under aberrant conditions vs. normal conditions, but inillustrative embodiments, bind with higher affinity under aberrantconditions than normal conditions. In some embodiments, thesepolypeptides can bind to their cognate antigen with a 10%, 20%, 25%,50%, 75%, 90%, 95% or 99% greater affinity under aberrant conditionsthan physiological (i.e. normal) conditions. In some embodiments, theASTRs identifying using methods provided herein do not bind to theircognate antigens under normal physiological conditions to any detectablelevel above background levels obtained using negative controls, such asnegative control antibodies.

The nucleotide sequence encoding a microenvironment restricted ASTRisolated by the method provided herein, can be determined by sequencingnucleotides of the collected cell expressing the microenvironmentrestricted antigen-specific targeting. This nucleotide sequenceinformation can then be used to make a microenvironment restrictedbiologic chimeric antigen receptor (MRB-CAR) by generating apolynucleotide that encodes a polypeptide comprising themicroenvironment restricted antigen-specific targeting region(MRB-ASTR), a transmembrane domain, and an intracellular activatingdomain. Microenvironment restricted antigen-specific targeting regionscan be cloned into a CAR construct expression system, which can be usedto generate recombinant lentiviruses that include the CAR in theirgenome, and then the recombinant lentiviruses can be used to transduce Tcells for testing for CAR-mediated tumor antigen expressing target cellkilling in a tumor-selective environment compared to physiologicconditions.

Conditions for Conditional Activity

In the methods provided herein, the activity of one or morepolypeptides, such as, for example, single chain antibodies, is screenedor tested under two different sets of conditions that simulate acondition or conditions in two different physiologic environments suchas, for example, a diseased microenvironment and the normal physiologiccondition of a non-diseased microenvironment. Typically, the conditionsare conditions that can be simulated or replicated in vitro. A set ofconditions can include one or more conditions to simulate amicroenvironment associated with a disease. Disease can alterintracellular and extracellular homeostasis. For example, the diseasedmicroenvironment can simulate one or more conditions in a tumormicroenvironment or a cancer microenvironment. Typically, the differenceor differences in activity under the two sets of conditions can resultin the conditional activity of the molecule. Thus, a molecule thatexhibits greater activity under the first set of conditions (e.g.simulating conditions in a tumor microenvironment) compared to thesecond set of conditions (e.g. simulating conditions in a normal ornon-diseased environment) is identified as a candidate molecule that ismicroenvironment restricted.

The two sets of conditions can be selected to vary by one or moreparameters that differ in two physiologic environments, such asdescribed herein or known to one of skill in the art, including but notlimited to chemical conditions, biological conditions, or physicalconditions. Parameters that can be varied between the two sets ofconditions can include one or more conditions selected from amongpressure, temperature, pH, ionic strength, osmotic pressure, osmolality,oxidative stress, turbidity, exposure to light (including UV, infraredor visible light), concentration of one or more solutes, such aselectrolytes, concentration of glucose, concentration of hyaluronan,concentration of lactic acid or lactate, concentration of albumin,levels of adenosine, levels of R-2-hydroxyglutarate, concentration ofpyruvate, concentration of oxygen, and/or presence of oxidants,reductants, or co-factors. By varying the electrolyte and buffer systemsin the calibration solutions, physiological conditions such as pH,buffer capacity, ionic environment, temperature, glucose concentration,and ionic strength can be adjusted to those of the biologicalenvironment to be simulated. The set of conditions that simulate anormal physiologic environment can be selected to be different from theset of conditions that simulate a diseased microenvironment, such as atumor microenvironment, by one or more conditions described herein.

For example, as discussed below, various parameters of the tumormicroenvironment differ compared to a non-tumor microenvironment,including, but not limited to, oxygen concentration, pressure, presenceof co-factors, pH, hyaluronan concentration, lactate concentration,albumin concentration, levels of adenosine, levels ofR-2-hydroxyglutarate, and pyruvate concentration. Any of theseparameters can be replicated in vitro to simulate one or more conditionsthat exist in a tumor or cancer environment compared to conditions thatexist in a non-tumor or a normal environment. The normal physiologicconditions that can be simulated include environments found in healthyor nondiseased tissue at any location of the body such as the GI tract,the skin, the vasculature, the blood, and extracellular matrix.Typically, in the assays herein, physiologic conditions can be simulatedin vitro by the choice of buffer that is used to assess the activity ofthe protein. For example, any one or more conditions of a diseasedmicroenvironment (such as a tumor microenvironment) and a non-diseasedenvironment can be simulated by differences in the assay buffer used toassess activity in the assay. Hence, in the methods herein to identify amicroenvironment restricted polypeptide, a component or components orcharacteristic or characteristics of an assay buffer are altered or madeto be different in a first assay to test activity under a firstcondition and in a second assay to test activity under a secondcondition. For example, as discussed herein, various parameters of thetumor microenvironment are different compared to a non-tumor environmentincluding, but not limited to, oxygen, pressure, presence of co-factors,pH, hyaluronan concentration (such as increased or decreased hyaluronanconcentration), lactate concentration (such as increased or decreasedlactate concentration), albumin concentration (such as increased ordecreased albumin concentration), levels of adenosine (such as increasedor decreased adenosine levels), levels of R-2-hydroxyglutarate (such asincreased or decreased R-2-hydroxyglutarate levels) and pyruvateconcentration (including increased or decreased pyruvate concentration).More specifically, conditions in a tumor microenvironment can includelower pH, higher concentrations of hyaluronan, higher concentrations oflactate and pyruvate, higher concentrations of albumin, increased levelsof adenosine, increased levels of R-2-hydroxyglutarate, hypoxia, lowerconcentration of glucose, and slightly higher temperature in comparisonwith non-tumor microenvironment. For example, a microenvironmentrestricted ASTR is virtually inactive at normal body temperature, but isactive at a higher temperature in a tumor microenvironment. In yetanother aspect, the microenvironment restricted antibody is less activein normal oxygenated blood, but more active under a less oxygenatedenvironment that exists in a tumor. In yet another aspect, themicroenvironment restricted antibody is less active in normalphysiological pH 7.2-7.8, but more active under an acidic pH 5.8-7.0, or6.0-6.8 that exists in a tumor microenvironment. For example, themicroenvironment restricted antibody is more active at a pH of 6.7 thanat pH 7.4. There are other conditions in the tumor microenvironmentknown to a person skilled in the field that may also be used as thecondition in the present invention under which the conditionally activeASTRs have different binding affinities. In vitro assay conditions thatmimic these in vivo tumor conditions are referred to herein as in vitrotumor surrogate assay conditions.

Any one or more of these conditions can be simulated in vitro by choiceof the particular assay buffer. The composition of the assay buffer thatsimulates a diseased microenvironment can be selected to be identical tothe composition of the assay buffer that simulate a normal environment,with the exception of one or more conditions known or described hereinthat is altered in the diseased microenvironment. Further, in screeningor identifying the activity of one or more polypeptides under twodifferent sets of conditions, generally the only conditions that arevaried in the assay relate to the buffer conditions simulating the invivo microenvironment. The other conditions of the assay, such as time,temperature and incubation conditions, can be the same for both sets ofconditions. Typically, the same base buffer is used in the set ofconditions that simulate a diseased microenvironment and conditions thatsimulate a normal microenvironment, but the design of the buffercomposition can be made to differ in one or more parameters such as pH,oxygen, pressure, presence of co-factors, pH, hyaluronan concentration(such as increased or decreased hyaluronan concentration), lactateconcentration (such as increased or decreased lactate concentration),albumin concentration (such as increased or decreased hyaluronanconcentration) and/or pyruvate concentration (including increased ordecreased pyruvate concentration). In the conditions that simulate adiseased microenvironment and the conditions that simulate a normalmicroenvironment, any base buffer known to one of skill in the art thatcan be used

Methods of Generating a Microenvironment Restricted Cell

The present disclosure provides a method of generating amicroenvironment restricted cell. The method generally involvesgenetically modifying a mammalian cell with an expression vector (e.g. aplasmid or a retroviral vector), or an RNA (e.g., in vitro transcribedRNA), including nucleotide sequences encoding microenvironmentrestricted CARs of the present disclosure. The genetically modified cellis microenvironment restricted in the presence of one or more targetantigens. The genetic modification can be carried out in vivo, in vitro,or ex vivo. The cell can be an immune cell (e.g., a T lymphocyte, aT-helper cell, or an NK cell), a stem cell, a progenitor cell, etc.

In some cases, the genetic modification is carried out ex vivo. Forexample, a T lymphocyte, a stem cell, a T-helper cell, or an NK cell isobtained from an individual; and the cell obtained from the individualis genetically modified to express a CAR of the present disclosure. Thegenetically modified cell is microenvironment restrictable in thepresence of one or more target antigens. In some cases, the geneticallymodified cell is activated ex vivo. In other cases, the geneticallymodified cell is introduced into an individual (e.g., the individualfrom whom the cell was obtained); and the genetically modified cell isactivated in vivo. For example, where the one or more target antigensare present on the surface of a cell in the individual, there is no needto administer the antigen. The genetically modified cell comes intocontact with the antigen present on the surface of a cell in theindividual and the genetically modified cell is activated. For example,where the genetically modified cell is a T lymphocyte, the geneticallymodified cell can exhibit cytotoxicity toward a cell that expresses theone or more target antigens on its surface to which the CAR binds.

Methods for Modulating MRB Car-Expressing T Cell and/or Nk Cell Activityby Changing pH

Provided herein in certain aspects, are methods for modulating bindingand resulting lysis/killing of a target cell by an MRB CAR-expressing Tcell and/or NK cell by causing a change or shift in pH within amicroenvironment that includes a target cell either within a targettissue or within one or more non-target (e.g. healthy/normal) tissues,by modulating binding of the MRB-CAR to its cognate antigen on a targetcell(s). Such methods typically include contacting a target cell, suchas a mammalian cell (e.g. a human cell) with an MRB CAR-expressing Tcell and/or NK cell in a microenvironment and then changing the pH ofthe microenvironment, either by decreasing or more typically increasingthe pH. The microenvironment can be a target microenvironment, forexample a tumor, or an off-target microenvironment, where off-targetbinding can cause side-effects. In some embodiments, such methods canprovide a transient reduction of tumor microenvironment sensitive CAR-Ttarget binding.

Accordingly, in one aspect, provided herein is a method for modulatingbinding of a microenvironment restricted biologic chimeric antigenreceptor (MRB-CAR)-expressing T cell or NK cell to a cell expressing acognate antigen of the MRB-CAR in a subject, that includes thefollowing:

-   -   a. introducing a T cell and/or NK cell comprising a nucleic acid        encoding the MRB-CAR into the subject, wherein after (and        optionally and/or during) the introducing, the T cell and/or the        NK cell comprising the nucleic acid encoding the MRB-CAR        expresses the MRB-CAR and binds to the cell expressing the        cognate antigen in the subject; and    -   b. administering a pharmacologic agent to the subject in        sufficient amount to increase blood pH and/or pH of a tissue        and/or pH of a microenvironment, wherein the administering is        performed before, during, or after the introducing, and wherein        the increased pH of the blood, the tissue, and/or the        microenvironment modulates binding of the MRB-CAR expressing T        cell and/or NK cell to the cell expressing the cognate antigen        in the blood, the tissue, or the microenvironment with the        increased pH.

The change/shift in pH in aspects that include a step of administering apH-modulating pharmacologic agent of the present disclosure can beaccomplished by exposing target or non-target cells/tissue to apH-modulating pharmacologic agent, such as by administering the pHmodulating pharmacologic agent to a subject. Non-limiting examples ofpH-modulating pharmacologic agents are provided herein. In certainaspects, provided herein is a pharmacologic agent for use in a methodfor modulating binding of an MRB-CAR to its cognate antigen or formodulating binding of an MRB CAR-expressing T cell and/or NK cell to acell that expresses its cognate antigen or for reducing or alleviatingon target off tumor toxicity in a subject. Such aspects in certainembodiments, relate to treating tumor growth, cancer, hyperplasia, orcell proliferative disorders.

In other aspects, provided herein is a use of a pH-modulatingpharmacologic agent for use in the manufacture of a medicament or a kitfor controlling binding of a genetically engineered T cell and/or NKcell to a target mammalian cell in a subject in vivo. In other aspects,provided herein is a kit that includes a container containing areplication incompetent recombinant retroviral particle, andinstructions for use thereof for performing a method for treating tumorgrowth, wherein the instructions instruct a method for controllingbinding of a T cell and/or NK cell to a target mammalian cell bymodulating pH. Such method can be any of the methods provided hereinthis section for modulating MRB CAR-expressing T cell and/or NK cellbinding and/or activity by changing pH. The container that contains therecombinant retroviral particles can be a tube, vial, well of a plate,or other vessel for storage of a recombinant retroviral particle and/ora pH-modulating pharmacologic agent. Any of these can be of industrialstrength and grade. The kit can include two or more containers incertain embodiments. One container/vessel can include the recombinantretroviral particles and another container/vessel can include apH-modulating pharmacologic agent. In such methods the pharmacologicagent is delivered/administered in sufficient amount to increase bloodpH and/or a tissue pH and/or a microenvironment pH to modulate bindingof the MRB-CAR of a modified/recombinant T cell and/or NK cellexpressing the MRB CAR, to its cognate antigen in the blood and/or thetissue with the increased pH. Non-limiting exemplary details areprovided herein for administering a pH modulating pharmacologic agent insufficient amount and for a sufficient time.

Target cells, whether on target or off target with respect to a tissue,can be contacted with a pH modulating agent, such as a pH modulatingpharmacologic agent, after introducing the MRB-CAR into a subject.Accordingly, exemplary aspects provided herein for modulating bindingand/or cytotoxic activity of an MRB CAR-expressing T cell, for examplefor alleviating on target off tumor activity and/or for inhibitingtarget cell proliferation, such as tumor cell proliferation, can includethe following steps:

-   -   a. introducing a T cell and/or NK cell comprising a nucleic acid        encoding an MRB-CAR into a subject wherein after the        introducing, the T cell and/or the NK cell comprising the        nucleic acid encoding the MRB-CAR expresses the MRB-CAR, and        optionally binds to the cell expressing the cognate antigen in        the subject; and    -   b. administering a pharmacologic agent to the subject in        sufficient amount to increase blood pH and/or a tissue pH and/or        a microenvironment pH to modulate binding of the MRB        CAR-expressing T cell and/or NK cell to cells expressing the        cognate antigen of the MRB CAR, in the blood, the tissue, or the        microenvironment with the increased pH. It will be understood        that depending on the specific method used to introduce the        nucleic acid encoding the MRB-CAR into the T cell and/or NK        cell, the T cell and/or NK cell may or may not express the        MRB-CAR before it is introduced into the subject. However, at        some timepoint after introduction into the subject, e.g. 2        hours, 4 hours, 8 hours, 12 hours, 1 day, 2 days, 4 days and/or        7 days, or longer, the T cell and/or NK cell that include the        nucleic acid encoding the MRB-CAR, express the MRB-CAR. Then        such cells typically bind to a target cell expressing the        cognate antigen for the MRB-CAR.

Methods provided herein for genetically modifying and optionallyexpanding lymphocytes of a subject can be used to introduce a nucleicacid sequence that encodes an MRB-CAR into the genome of a T cell and/orNK cell of the subject to produce an T cell and/or NK cell capable ofexpressing the MRB CAR, and then to introduce the T cell and/or NK cellcapable of expressing the MRB CAR into the subject, wherein afterintroducing the T cell and/or NK cell expresses the MRB CAR in order tocontact the MRB-CAR with a target cells/tissue. The present disclosureprovides details of how to perform such methods, along with variousalternatives for modifying and expanding lymphocytes, any of which canbe used in aspects of the disclosure that include changing pH tomodulate binding of an MRB CAR-expressing T cell and/or NK cell to atarget cell expressing a cognate antigen for the MRB-CAR.

Such methods for genetically modifying and expanding lymphocytestypically involve contacting T cells and/or NK cells, which can beresting cells in illustrative embodiments, with a replicationincompetent recombinant retroviral particle to transduce the T cellsand/or NK cells. Such contacting typically occurs ex vivo after removingthe lymphocytes from the subject. The T cells and/or NK cells are thenintroduced/reintroduced into the subject, typically from whom they wereremoved. The replication incompetent recombinant retroviral particleincludes a genome with a polynucleotide that encodes the MRB-CAR. Manyalternative embodiments and further details regarding such a replicationincompetent recombinant retroviral particle are provided in othersections herein and can be used in methods provided herein forregulating binding and resulting lysis/killing of MRB-CARs by modulatingpH in a microenvironment of a cell expressing a cognate targetpolypeptide recognized by the MRB-CAR in a pH-dependent manner.

Particularly illustrative aspects that include such combinations caninclude other elements for regulating binding, cell killing activity,and/or survival of MRB CAR-expressing T cells that are provided hereinin other sections. Such control elements that can be combined with MRBCAR-expression in methods that include a change in pH as well as otherembodiments provided herein, include riboswitches and eliminationdomains, Thus, the combination of such methods and compositions providedherein, form a powerful multi-faceted approach to assuring safety of asubject after administration of CAR-expressing T cells, including MRBCAR-expressing T cells, to a subject.

Such methods for modulating binding of a target cell by an MRBCAR-expressing T cell and/or NK cell can be used, for example, to reduceon target, off-tumor toxicity by increasing the pH of blood and/or anon-tumor tissue(s) within the subject. For example, in a situationwhere a “normal” tissue pH within a subject becomes transiently lower, apH modulating agent can be delivered in a manner where pH of the normaltissue is increased while pH of the tumor remains lower and still at apH where the MRB-CAR-expressing T cell and/or NK cell binds a targettumor cell. In these embodiments, the pH modulating agent can bedelivered at a lower concentration or in a targeted manner to the normaltissue.

In some embodiments, this can be accomplished while allowing the pHwithin the tumor microenvironment to remain low enough for an MRB-CAR Tcell and/or NK cell to bind to its cognate target-expressing cellswithin the tumor. In illustrative aspects of methods provided herein,the pH of a tissue remains at a pH under which an MRB CAR-expressing Tcell and/or NK cell binds its target for a period of time sufficient fora MRB CAR-expressing T cell and/or NK cell to contact and bind to a cellexpressing its cognate antigen (e.g. 2, 4, 8, 12, or 24 hours, or 2, 4,7, 14, 28, or 30 days, or 1, 2, 3, 4, 5, 6, 12, 24 months, or longer),and then the pH is shifted/changed, for example by increasing the pH ofthe tissue to such a magnitude as to affect binding of the MRBCAR-expressing T cell and/or NK cell to a target cell.

Accordingly, provided herein, in one aspect, is a method for transientreduction of tumor microenvironment sensitive CAR-T cell target bindingthrough pharmacologic modification of vascular and tissue pH. The targetbinding portions of the tumor microenvironment sensitive CAR-T cell withdifferent binding in different conditions are also referred to asmicroenvironment restricted biologic, microenvironment restricted,microenvironmentally controlled, or conditionally active and can referto the entire CAR or any target binding domain thereof, for example, anASTR, scFv, or scFvFc. These microenvironmentally controlled ASTRs inCAR-T cells provide an additional level of protection against on-targetoff tumor toxicity, requiring tumor local environmental conditions toenable T cell engagement. While attractive for some monoclonal antibodytherapies, adoptive cellular therapy may create local environments thatare transiently permissive for their CAR-T targets. For example, CAR-Tcells activated in tissues with a low pH may further reduce the pH ofthe microenvironment, depending on cytoplasmic domains present in theCAR construct. In other instances, cytokine release syndrome and othermorbidity associated with adoptive cellular therapy may result in lossof the bicarbonate buffering capacity of blood, leading to lacticacidosis. It has been established that adoptive cellular therapiesadministered by intravenous infusion result in temporary pulmonaryentrapment. For some cellular therapies, infusion rate requires constantmonitoring of dissolved oxygen (Fischer et al. Stem Cells Dev. 2009June; 18(5): 683-691). The extent of pulmonary entrapment is dependentupon cell size, activation state, cell dose, and infusion rate. Cruz etal (Cytotherapy. 2010 October; 12(6): 743-749) report the adversefindings from over 300 T cell infusions, that low doses and slowinfusion may reduce pulmonary entrapment. However, with certain highpotency CAR-T cells, targets present even in low levels on lungendothelium, such as Her2 (Morgan et al. Mol Ther. 2010 April; 18(4):843-851), can result in immediate toxicity that cannot be controlled,and results in rapid patient deterioration due to the initial high CAR-Tcellular concentration in the lung following infusion and the presenceof the T cell target in these tissues. In other cases, the presence of Tcell targets in other off target tissues such as bile duct may create ontarget off tumor toxicities that cannot be controlled (Lamers Mol Ther.2013 April; 21(4):904-12) and result in severe organ toxicity beforeother agents such as steroids or cell elimination epitopes can beutilized. While venous and arterial plasma have strong bufferingcapacity against acidosis, conditions of respiratory acidosis, shock,metabolic acidosis and ischemic acidosis can occur in patients withcancer treated with adoptive cellular therapy.

In some aspects provided herein, the binding of an MRB-CAR in a subjectcan be modulated by administering a pharmacologic agent to the subjectto increase or decrease the pH of the blood, a tissue and/or amicroenvironment. In some aspects, on-target off tumor toxicity can bealleviated in a subject by administering a pharmacologic agent to thesubject to increase or decrease the blood pH and/or the pH of a tissueand/or the pH of a microenvironment. In some aspects, the binding of a Tcell and/or NK cell to a target mammalian cell can be controlled byintroducing a pharmacologic agent to increase or decrease the blood pHand/or the pH of a tissue and/or the pH of a microenvironment. In someaspects, the binding of a genetically engineered T cell and/or NK cellto a target mammalian cell in a subject in vivo can be controlled byadministering a pH-modulating pharmacologic agent to the subject. Inillustrative embodiments, the pharmacologic agent can increase the bloodpH and/or the pH of a tissue and/or the pH of a microenvironment. Insome embodiments, the microenvironment can be an in vivomicroenvironment. In illustrative embodiments, the microenvironment canbe a tumor microenvironment. In some embodiments, the microenvironmentcan include a target mammalian cell, wherein the target mammalian cellexpressed the target antigen on its surface. In some embodiments,administering a pharmacologic agent to a subject can increase the pH ofblood, a tissue, and/or a microenvironment from a pH of less than 5.8,5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or 6.9 to a pH of atleast 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6, whereinthe pH of the blood, tissue, and/or microenvironment is lower beforeadministering the pharmacologic agent than after administering thepharmacologic agent. In some embodiments, administering a pharmacologicagent to a subject can decrease the pH of blood, a tissue, or amicroenvironment from a pH of more than 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,7.2, 7.3, 7.4, 7.5, or 7.6 to a pH of less than 5.8, 5.9, 6.0, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0, wherein the pH of the blood,tissue, and/or microenvironment is higher before administering thepharmacologic agent than after administering the pharmacologic agent. Insome embodiments, administering a pharmacologic agent to a subject cancause a pH shift in the subject in the blood, a tissue, and/or amicroenvironment. In some embodiments, the pH shift can be at least 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, or 1.8 pH units in either direction, i.e. an increase ordecrease in pH after administering the pharmacologic agent relative tothe pH before administering the pharmacologic agent. In illustrativeembodiments, the pH shift is an increase in pH.

The MRB-CARs of the present disclosure can have reduced binding to itscognate antigen at one pH than at a different pH. In illustrativeembodiments where illustrative pH values for differential binding of anMRB-CAR are not already provided in the broadest aspect andalternatively for other embodiments in place of those values for suchaspects, the MRB-CAR can have reduced binding at a higher pH than at alower pH. For example, the MRB-CAR can have reduced binding to itscognate antigen at a pH above 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5 than at apH below 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. In other embodiments, theMRB-CAR can have reduced binding at a higher pH than at a lower pH. Forexample, the MRB-CAR can have reduced binding to its cognate antigen ata pH below 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0 than at a pH above 7.0,7.1, 7.2, 7.3, 7.4, or 7.5. In some illustrative embodiments, theMRB-CAR exhibits increased binding at a pH of 6.5 to 6.7 compared to pH7.4 to 7.6. In other illustrative embodiments, the MRB-CAR exhibitsincreased binding at a pH of 6.7 compared to a pH of 7.4. In otherembodiments, the MRB-CAR exhibits increased binding in the pH of a tumorcompared to the pH of blood. In some embodiments, the MRB-CAR caninclude an antigen-specific targeting region, a stalk, and anintracellular activating domain. In some embodiments, the MRB-CAR canalso include a co-stimulatory domain. In some embodiments, the MRB-CARcan bind to a tumor associated antigen.

In methods that include modulating the pH of the blood, a tissue, or amicroenvironment, the pH of the microenvironment can be increased from apH below 7.0 to a pH above 7.0. For example, the pH can be increasedfrom a pH below 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0 to a pH above 7.0,7.1, 7.2, 7.3, or 7.4. In some embodiments, the MRB-CAR can bind to thecognate antigen at the increased pH but not a pH of the microenvironmentbefore introducing the pharmacologic agent. In certain embodiments, thepH can be increased from below 7.0 to a pH of 7.1 to 8.0 or to a pH of7.1 to 7.8 or to a pH of 7.2 to 7.8 or a pH of 7.2 to 7.6 or a pH of 7.3to 7.6 or to a pH of 7.4 to 7.8 or to a pH of 7.4 to 7.6. Such anincrease in pH can occur for less than 1, 2, 4, 6, 8, 12, or 24 hours orfor more than 1, 2, 4, 6, 8, 12 or 24 hours depending on the type anddose of pharmacologic agent administered. In certain embodiments, thepharmacologic agent is administered such that the pH remains above 7.0,7.1, 7.2, 7.3, 7.4, or 7.5; or between 7.0, 7.1, 7.2, 7.3 on the low endof the range and 7.4, 7.5, 7.6, 7.7, or 7.8 on the high end of therange, in the target tissue, such as a tumor, and for example in atleast a surface of a target tissue (e.g. tumor) microenvironment, in atleast a portion of a target tissue (e.g. tumor) microenvironment, and inillustrative embodiments throughout a target tissue (e.g. tumor)microenvironment. The microenvironment can be an in vivomicroenvironment, such as a tumor, a tissue, a non-tumor tissue, anormal tissue, or a tissue that has undergone a transient shift in pH.For example, tissues that typically undergo transient shifts in pHinclude a muscle tissue in anaerobic conditions or muscle tissueundergoing exercise or an inflamed tissue or a tissue experiencinginflammation. In some embodiments that include a target mammalian cell,the target mammalian cell can be a tumor cell or a non-tumor or normalcell.

In some aspects, methods for transiently increasing vascular pH toreduce affinity of microenvironmentally controlled MRB-CARs for theirantigens are provided. A 0.4U shift in blood pH can reduce the affinityof certain scFvs that form a portion of an MRB-CAR, for their cognateantigen by greater than 10-fold. In some embodiments, therapeutic pHcontrol can be achieved via IV or oral administration routes of variouspharmacologic agents. For example, in some embodiments, inactivation ofbinding affinity can be achieved with bicarbonate or sodium bicarbonate.In other embodiments, Tris-hydroxymethyl aminomethane (also known astromethamine, trometamol, and THAM) and/or Carbicarb™ (an equimolarhypertonic solution of sodium bicarbonate and sodium carbonate) can beutilized to increase the pH of the blood in a sufficient amount toalleviate on-target off tumor toxicities. In still other embodiments,small molecule proton pump inhibitors can be utilized to increase bloodpH and/or tissue pH in a sufficient amount to alleviate on-target offtumor toxicities. Proton pump inhibitors that can be used in methodsthat include modulating pH include, but are not limited to, esomeprazole(Nexium), esomeprazole and naproxen (Vimovo), lansoprazole (Prevacid),omeprazole (Prilosec and Zegerid), and rabeprazole (Aciphex).Administration of proton pump inhibitors can be used effectively overlonger time periods to modulate the binding affinity of the antigenbiding domain to its cognate antigen for days, weeks, months, or years.In other embodiments, the affinity of the antigen binding domain for itscognate antigen can be modulated by altering the blood pH and/or tissuepH by controlling the transcription, translation, membrane expression,and stability of transporters and pumps. Examples of such transportersand pumps whose altered expression can be to modulate pH include, butare not limited to, proton pumps, members of the sodium proton exchangefamily (NHE), bicarbonate transporter family (BCT), and monocarboxylatetransporter family.

In certain embodiments, a pH-modulating pharmacologic agent, such as,for example, bicarbonate, THAM, or Caricarb™ are administered prior toor concurrent with infusion of a patient's CAR-T cells expressingmicroenvironment restricted biologic ASTRs (e.g. scFvs or scFvFcs). Suchtreatment will alleviate the immediate cytoxicity that is otherwiseassociated with the temporary pulmonary entrapment of CAR-T cellinfusions. Accordingly, in certain aspects provided herein is a methodfor reducing cytotoxicity caused to normal, healthy tissue of a subjectby administering a pharmacologic agent to the subject in sufficientamount to increase blood pH and/or a tissue pH and/or a microenvironmentpH; and either concomitantly or subsequently (e.g. 1, 2, 4, 6, 8, 12, or24 hours, or 1, 2, 3, 4, or 7 days later) introducing an MRBCAR-expressing T cell or NK cell into the subject. In certainembodiments, at a target time after such introducing (e.g. 1, 2, 4, 6,8, 12, or 24 hours, or 1, 2, 3, 4, or 7 days later), administration ofthe pharmacologic agent is terminated for a period of time orindefinitely, in order to change the pH of the blood, a tissue, or amicroenvironment of the subject and modulate binding/activity of the MRBCAR-expressing T cell.

Various effective dosing regimens for administering the pharmacologicagents capable of modulating pH (e.g. increasing blood pH and/or atissue pH and/or the pH of a microenvironment in a subject) can be used,as will be understood by a skilled artisan. Herein, administering canrefer to giving a pharmacologic agent to a subject including injecting apharmacologic agent through an IV into a subject or providing an oraldose of a pharmacologic agent to a subject or a subject taking apharmacologic agent. The pharmacologic agents can be administered to thesubject or patient for various lengths of time, for example, at least 1,2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 weeks; 3, 4,5, 6, 7, 8, 9, 10, 12, 15, or 18 months; or 2, 2.5, 3, 3.5, 4, 4.5, or 5years or indefinitely. In some embodiments, the pharmacologic agent canbe bicarbonate, sodium bicarbonate (NaHCO₃), or a solution of sodiumbicarbonate and sodium carbonate and a parenteral or IV dosage can be:0.2×weight of subject (kg)×base deficit of the subject; HCO₃ (mEq)required=0.5×weight (kg)×[24− serum HCO₃ (mEq/L)]; or 2 to 5 mEq/kg IVinfusion over 4 to 8 hours. In some embodiments, standard dosingregimens of bicarbonate, sodium bicarbonate, or a solution of sodiumbicarbonate can be used depending on the severity of the acidosis. Forexample, 50 to 150 mEq bicarbonate diluted in 1 L of 5% dextrose inwater can be administered via IV at a rate of 1 to 1.5 L/hour. Inanother non-limiting example, 90 to 180 mEq bicarbonate diluted in 1 Lof 5% dextrose in water can be administered via IV at a rate of 1 to 1.5L/hour. In some embodiments where the pharmacologic agent is bicarbonateor sodium bicarbonate (NaHCO₃), an enteral or oral dosage can be, forexample, 325 to 2000 mg sodium bicarbonate given to a subject 1 to 4times/day.

In some embodiments, the pharmacologic agent can be tris-hydroxymethylaminomethane (also known as tromethamine, trometamol, and THAM) and aparenteral or IV dosage can be estimated as: Tromethamine solution (mLof 0.3 M) required=Body Weight (kg) x Base Deficit (mEq/liter)×1.1. Insome embodiments, the IV dosage of tris-hydroxymethyl aminomethane canbe estimated from the buffer base deficit of the extracellular fluid inmEq/L as determined by means of the Siggaard-Andersen nomogram. In someembodiments, the initial dose can be 500 ml (150 mEq) oftris-hydroxymethyl aminomethane injected by slow IV infusion with up to1000 mL, wherein the maximum dose is 500 mg/kg (227 mg/lb) over a periodof not less than one hour.

In some embodiments, the pharmacologic agent can be a small moleculeproton pump inhibitor and can be administered for extended treatmentlengths. For example, the small molecule proton pump inhibitor can beadministered for at least 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or 11 weeks; 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 18 months; or2, 2.5, 3, 3.5, 4, 4.5, or 5 years or indefinitely. In some embodiments,the proton pump inhibitor can be esomeprazole (Nexium) and 20 mg or 40mg esomeprazole can be administered orally once or twice daily. In someembodiments, the proton pump inhibitor can be a combination ofesomeprazole and naproxen (Vimovo) and 20 mg esomeprazole with 375 or500 mg naproxen can be administered orally twice daily. In someembodiments, the proton pump inhibitor can be lansoprazole (Prevacid)and 15, 30, or 60 mg lansoprazole can be administered orally once ortwice daily. In some embodiments, lansoprazole can be administered by IVwith 30 mg lansoprazole injected over 30 minutes once daily for up to 7days. The subject can then switch to oral lansoprazole and continuetreatment. In some embodiments, the proton pump inhibitor can beomeprazole (Prilosec and Zegerid) and 10, 20, or 40 mg omeprazole can beadministered orally once or twice daily. In some embodiments, the protonpump inhibitor can be rabeprazole (Aciphex) and 20 or 60 mg rabeprazolecan be administered orally once or twice daily or 100 mg rabeprazole canbe administered orally once daily. In any of the embodiments disclosedherein, the pharmacologic agents can be used in combination with eachother.

In any of the embodiments disclosed herein, the pH of the blood, atissue, and/or a microenvironment of a subject can be measured before,during, or after the administration of a pharmacologic agent. In someembodiments, the decision to administer or to continue to administer, toa subject the pharmacologic agent to increase or decrease the pH can bebased on the pH measurement of the blood, a tissue, and/or amicroenvironment of the subject. Methods to measure the blood pH and/orbicarbonate levels of the blood of a subject are well-known in the art.In some embodiments, positron emission tomography (PET), magneticresonance spectroscopy (MRS), magnetic resonance imaging (MRI), andoptical imaging can be used to measure in vivo pH in microenvironments,for example, in tumors (for details of measuring tumor pH, see: Zhang X,Lin Y, Gillies RJ. Tumor pH and its measurement. J Nucl Med. 2010August; 51(8):1167-70).

In another aspect, provided herein is a method for alleviating on targetoff tumor toxicity in a subject, that includes the following:

-   -   a. introducing a polynucleotide encoding an microenvironment        restricted biologic chimeric antigen receptor (MRB-CAR) into a T        cell or NK cell of the subject to produce a T cell and/or NK        cell capable of expressing the MRB-CAR;    -   b. introducing the T cell and/or NK cell capable of expressing        the MRB-CAR into the subject, wherein the T cell and/or NK cell        express the MRB-CAR in the subject; and    -   c. administering a pharmacologic agent to the subject in        sufficient amount to increase blood pH and/or pH of a tissue        and/or pH of a microenvironment to modulate binding of the        MRB-CAR to its cognate antigen in the blood, the tissue, and/or        the microenvironment with the increased pH, thereby alleviating        on target off tumor toxicity in the subject.

In the introducing step, the T cell or NK cell is capable of expressingthe MRB-CAR because it is genetically modified to contain the nucleicacid that encodes the MRB-CAR. This genetic modification can be thepresence of the MRB-CAR coding sequence on a vector that has beenintroduced inside the T cell or NK cell by transfection or transduction.In illustrative embodiments the nucleic acid encoding the MRB-CAR isintegrated into the genome of the T cell or NK cell.

It is envisioned that various methods known in the art for introducing apolynucleotide into a T cell and/or NK cell could be used with methodsprovided herein for aspects that include changing pH to affect bindingof an MRB-CAR T cell or NK cell to its cognate antigen on a cell usingan agent such as a pH-modulating pharmacologic agent (sometimes referredto herein as “pH Switch aspects”). Typically, a vector, in illustrativeexamples an expression vector, is used to deliver the polynucleotide.Such vectors can include various vectors known in the art for deliverynucleic acids to T cells and/or NK cells. Illustrative aspects of theinvention utilize retroviral vectors and retroviral particles, and insome particularly illustrative embodiments lentiviral vectors and inillustrative embodiments, recombinant lentiviral particles.

Other suitable expression vectors can be used in pH switch aspectsprovided herein. Such expression vectors include, but are not limitedto, viral vectors (e.g. viral vectors based on vaccinia virus;poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li andDavidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:10881097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO95/11984 and WO 95/00655); adeno-associated virus (see, e.g., Ali etal., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921,1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997;Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastavain WO 93/09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelsonet al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; or a retroviral vector (e.g.,Murine Leukemia Virus, spleen necrosis virus, and vectors derived fromretroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avianleukosis virus, human immunodeficiency virus, myeloproliferative sarcomavirus, and mammary tumor virus), for example a gamma retrovirus; orhuman immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:1031923, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); and the like.

In some embodiments, DNA-containing viral particles are utilized insteadof recombinant retroviral particles. Such viral particles can beadenoviruses, adeno-associated viruses, herpesviruses,cytomegaloviruses, poxviruses, avipox viruses, influenza viruses,vesicular stomatitis virus (VSV), or Sindbis virus. A skilled artisanwill appreciate how to modify the methods disclosed herein for use withdifferent viruses and retroviruses, or retroviral particles. Where viralparticles are used that include a DNA genome, a skilled artisan willappreciate that functional units can be included in such genomes toinduce integration of all or a portion of the DNA genome of the viralparticle into the genome of a T cell and/or NK cell transduced with suchvirus. Alternatively, functional DNA can be delivered to a T cell and/orNK cell that is expressed in the cell but is not integrated into thegenome of the T cell and/or NK cell.

In illustrative embodiments, the vector used in a pH switch aspect ofthe present disclosure is a recombinant retroviral particle and incertain embodiments, a recombinant lentiviral particle. Such retroviralparticle typically includes a retroviral genome within a capsid which islocated within a viral envelope. The present disclosure in varioussections herein, provide various embodiments of recombinant retroviralparticles that disclose elements that can be included on the surface orwithin, and/or in the genome of a recombinant retroviral particle. Anyof these recombinant retroviral particle embodiments can be used in thepH switch aspects provided herein.

Inhibitory RNA Molecules

In certain embodiments, methods provided herein for the presentdisclosure include inhibiting expression of one or more endogenous genesexpressed in T cells and/or NK cells. Methods provided herein illustratethe ability to make recombinant retroviral particles that express one ormore, and in illustrative embodiments two or more, inhibitory RNAmolecules, such as for example, a miRNA or shRNA, that can be used forsuch methods. In fact, the methods provided herein illustrate that suchinhibitory RNA molecules can be encoded within introns, including forexample, an Ef1a intron. This takes advantage of the present teachingsof methods to maximize the functional elements that can be included in apackageable retroviral genome to overcome shortcomings of priorteachings and maximize the effectiveness of such recombinant retroviralparticles in adoptive T cell therapy.

In some embodiments, the inhibitory RNA molecule includes a 5′ strandand a 3′ strand (in some examples, sense strand and antisense strand)that are partially or fully complementary to one another such that thetwo strands are capable of forming a 18-25 nucleotide RNA duplex withina cellular environment. The 5′ strand can be 18, 19, 20, 21, 22, 23, 24,or 25 nucleotides in length, and the 3′ strand can be 18, 19, 20, 21,22, 23, 24, or 25 nucleotides in length. The 5′ strand and the 3′ strandcan be the same or different lengths, and the RNA duplex can include oneor more mismatches. Alternatively, the RNA duplex has no mismatches.

The inhibitory RNA molecules included in compositions and methodsprovided herein, in certain illustrative examples, do not exist and/orare not expressed naturally in T cells into whose genome they areinserted. In some embodiments, the inhibitory RNA molecule is a miRNA oran shRNA. In some embodiments, where reference is made herein or inpriority filings, to a nucleic acid encoding an siRNA, especially in acontext where the nucleic acid is part of a genome, it will beunderstood that such nucleic acid is capable of forming an siRNAprecursor such as miRNA or shRNA in a cell that is processed by DICER toform a double stranded RNA that typically interacts with, or becomespart of a RISK complex. In some embodiments, an inhibitory molecule ofan embodiment of the present disclosure is a precursor of a miRNA, suchas for example, a Pri-miRNA or a Pre-miRNA, or a precursor of an shRNA.In some embodiments, the miRNA or shRNA are artificially derived (i.e.artificial miRNAs or siRNAs). In other embodiments, the inhibitory RNAmolecule is a dsRNA (either transcribed or artificially introduced) thatis processed into an siRNA or the siRNA itself. In some embodiments, themiRNA or shRNA has a sequence that is not found in nature, or has atleast one functional segment that is not found in nature, or has acombination of functional segments that are not found in nature.

In some embodiments, inhibitory RNA molecules are positioned in thefirst nucleic acid molecule in a series or multiplex arrangement suchthat multiple miRNA sequences are simultaneously expressed from a singlepolycistronic miRNA transcript. In some embodiments, the inhibitory RNAmolecules can be adjoined to one another either directly or indirectlyby non-functional linker sequence(s). The linker sequence in someembodiments, is between 5 and 120 nucleotides in length, and in someembodiments can be between 10 and 40 nucleotides in length, asnon-limiting examples. In illustrative embodiments the first nucleicacid sequence encoding one or more (e.g. two or more) inhibitory RNAsand the second nucleic acid sequence encoding a CAR (e.g. an MRB-CAR)are operably linked to a promoter that is active constitutively or thatcan be induced in a T cell or NK cell. As such, the inhibitory RNAmolecule(s) (e.g. miRNAs) as well as the CAR are expressed in apolycistronic manner. Additionally, functional sequences can beexpressed from the same transcript. For example, any of thelymphoproliferative elements provided herein that are not inhibitory RNAmolecules, can be expressed from the same transcript as the CAR and theone or more (e.g. two or more) inhibitory RNA molecules.

In some embodiments, the inhibitory RNA molecule is a naturallyoccurring miRNA such as but not limited to miR-155. Alternatively,artificial miRNAs can be produced in which sequences capable of forminga hybridizing/complementary stem structure and directed against a targetRNA, are placed in a miRNA framework that includes microRNA flankingsequences for microRNA processing and a loop, which can optionally bederived from the same naturally occurring miRNA as the flankingsequences, between the stem sequences. Thus, in some embodiments, aninhibitory RNA molecule includes from 5′ to 3′ orientation: a 5′microRNA flanking sequence, a 5′ stem, a loop, a 3′ stem that ispartially or fully complementary to said 5′ stem, and a 3′ microRNAflanking sequence. In some embodiments, the 5′ stem (also called a 5′arm herein) is 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.In some embodiments, the 3′ stem (also called a 3′ arm herein) is 18,19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In someembodiments, the loop is 3 to 40, 10 to 40, 20 to 40, or 20 to 30nucleotides in length, and in illustrative embodiments the loop can be18, 19, 20, 21, or 22 nucleotides in length. In some embodiments, onestem is two nucleotides longer than the other stem. The longer stem canbe the 5′ or the 3′ stem.

In some embodiments, the 5′ microRNA flanking sequence, 3′ microRNAflanking sequence, or both, are derived from a naturally occurringmiRNA, such as but not limited to miR-155, miR-30, miR-17-92, miR-122,and miR-21. In certain embodiments, the 5′ microRNA flanking sequence,3′ microRNA flanking sequence, or both, are derived from a miR-155, suchas, e.g., the miR-155 from Mus musculus or Homo sapiens. Inserting asynthetic miRNA stem-loop into a miR-155 framework (i.e. the 5′ microRNAflanking sequence, the 3′ microRNA flanking sequence, and the loopbetween the miRNA 5′ and 3′ stems) is known to one of ordinary skill inthe art (Chung, K. et al. 2006. Nucleic Acids Research. 34(7):e53; U.S.Pat. No. 7,387,896). The SIBR (synthetic inhibitory BIC-derived RNA)sequence (Chung et al. 2006 supra), for example, has a 5′ microRNAflanking sequence consisting of nucleotides 134-161 (SEQ ID NO:256) ofthe Mus musculus BIC noncoding mRNA (Genbank ID AY096003.1) and a 3′microRNA flanking sequence consisting of nucleotides 223-283 of the Musmusculus BIC noncoding mRNA (Genbank ID AY096003.1). In one study, theSIBR sequence was modified (eSIBR) to enhance expression of miRNAs(Fowler, D. K. et al. 2015. Nucleic acids Research 44(5):e48). In someembodiments of the present disclosure, miRNAs can be placed in the SIBRor eSIBR miR-155 framework. In illustrative embodiments herein, miRNAsare placed in a miR-155 framework that includes the 5′ microRNA flankingsequence of miR-155 represented by SEQ ID NO:256, the 3′ microRNAflanking sequence represented by SEQ ID NO:260 (nucleotides 221-265 ofthe Mus musculus BIC noncoding mRNA); and a modified miR-155 loop (SEQID NO:258). Thus, in some embodiments, the 5′ microRNA flanking sequenceof miR-155 is SEQ ID NO:256 or a functional variant thereof, such as,for example, a sequence that is the same length as SEQ ID NO:256, or95%, 90%, 85%, 80%, 75%, or 50% as long as SEQ ID NO: 256 or is 100nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 55nucleotides or less, 50 nucleotides or less, 45 nucleotides or less, 40nucleotides or less, 35 nucleotides or less, 30 nucleotides or less, or25 nucleotides or less; and is at least 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% identical to SEQ ID NO:256. In some embodiments,the 3′ microRNA flanking sequence of miR-155 is SEQ ID NO:260 or afunctional variant thereof, such as, for example, the same length as SEQID NO:260, or 95%, 90%, 85%, 80%, 75%, or 50% as long as SEQ ID NO: 260or is a sequence that is 100 nucleotides or less, 95 nucleotides orless, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides orless, 75 nucleotides or less, 70 nucleotides or less, 65 nucleotides orless, 60 nucleotides or less, 55 nucleotides or less, 50 nucleotides orless, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides orless, 30 nucleotides or less, or 25 nucleotides or less; and is at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ IDNO:260. However, any known microRNA framework that is functional toprovide proper processing within a cell of miRNAs inserted therein toform mature miRNA capable of inhibiting expression of a target mRNA towhich they bind, is contemplated within the present disclosure.

In some embodiments, at least one, at least two, at least three, or atleast four of the inhibitory RNA molecules encoded by a nucleic acidsequence in a polynucleotide of a replication incompetent recombinantretroviral particle has the following arrangement in the 5′ to 3′orientation: a 5′ microRNA flanking sequence, a 5′ stem, a loop, a 3′stem that is partially or fully complementary to said 5′ stem, and a 3′microRNA flanking sequence. In some embodiments, all of the inhibitoryRNA molecules have the following arrangement in the 5′ to 3′orientation: a 5′ microRNA flanking sequence, a 5′ stem, a loop, a 3′stem that is partially or fully complementary to said 5′ stem, and a 3′microRNA flanking sequence. As disclosed herein, the inhibitory RNAmolecules can be separated by one or more linker sequences, which insome embodiments have no function except to act as spacers betweeninhibitory RNA molecules.

In some embodiments, where two or more inhibitory RNA molecules (in someexamples, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 inhibitory RNA molecules) areincluded, these inhibitory RNA molecules are directed against the sameor different RNA targets (such as e.g. mRNAs transcribed from genes ofinterest). In illustrative embodiments, between 2 and 10, 2 and 8, 2 and6, 2 and 5, 3 and 5, or 3 and 6 inhibitory RNA molecules are included inthe first nucleic acid sequence. In an illustrative embodiment, fourinhibitory RNA molecules are included in the first nucleic acidsequence.

In some embodiments, the RNA targets are mRNAs transcribed from genesthat are expressed by T cells such as but not limited to PD-1 (preventinactivation); CTLA4 (prevent inactivation); TCRα (safety—preventautoimmunity); TCRb (safety—prevent autoimmunity); CD3Z (safety—preventautoimmunity); SOCS1 (prevent inactivation); SMAD2 (preventinactivation); a miR-155 target (promote activation); IFN gamma (reduceCRS); cCBL (prolong signaling); TRAIL2 (prevent death); PP2A (prolongsignaling); ABCG1 (increase cholesterol microdomain content by limitingclearance of cholesterol). In illustrative examples, miRNAs insertedinto the genome of T cells in methods provided herein, are directed attargets such that proliferation of the T cells is induced and/orenhanced and/or apoptosis is suppressed.

In some embodiments, the RNA targets include mRNAs that encodecomponents of the T cell receptor (TCR) complex. Such components caninclude components for generation and/or formation of a T cell receptorcomplex and/or components for proper functioning of a T cell receptorcomplex. Accordingly, in one embodiment at least one of the two or moreof inhibitory RNA molecules causes a decrease in the formation and/orfunction of a TCR complex, in illustrative embodiments one or moreendogenous TCR complexes of a T cell. The T cell receptor complexincludes TCRα, TCRb, CD3d, CD3e, CD3g, and CD3z. It is known that thereis a complex interdependency of these components such that a decrease inthe expression of any one subunit will result in a decrease in theexpression and function of the complex. Accordingly, in one embodimentthe RNA target is an mRNA expressing one or more of TCRα, TCRb, CD3d,CD3e, CD3g, and CD3z endogenous to a transduced T cell. In certainembodiments, the RNA target is mRNA transcribed from the endogenous TCRαor TCRβ gene of the T cell whose genome comprises the first nucleic acidsequence encoding the one or more miRNAs. In illustrative embodiments,the RNA target is mRNA transcribed from the TCRα gene. In certainembodiments, inhibitory RNA molecules directed against mRNAs transcribedfrom target genes with similar expected utilities can be combined. Inother embodiments, inhibitory RNA molecules directed against targetmRNAs transcribed from target genes with complementary utilities can becombined. In some embodiments, the two or more inhibitory RNA moleculesare directed against the mRNAs transcribed from the target genes CD3Z,PD1, SOCS1, and/or IFN gamma.

In some embodiments provided herein, the two or more inhibitory RNAmolecules can be delivered in a single intron, such as but not limitedto EF1-aa intron A. Intron sequences that can be used to harbor miRNAsfor the present disclosure include any intron that is processed within aT cell. As indicated herein, one advantage of such an arrangement isthat this helps to maximize the ability to include miRNA sequenceswithin the size constraints of a retroviral genome used to deliver suchsequences to a T cell in methods provided herein. This is especiallytrue where an intron of the first nucleic acid sequence includes all ora portion of a promoter sequence used to express that intron, a CARsequence, and other functional sequences provided herein, such aslymphoproliferative element(s) that are not inhibitory RNA molecules, ina polycistronic manner. Sequence requirements for introns are known inthe art. In some embodiments, such intron processing is operably linkedto a riboswitch, such as any riboswitch disclosed herein. Thus, theriboswitch can provide a regulatory element for control of expression ofthe one or more miRNA sequences on the first nucleic acid sequence.Accordingly, in illustrative embodiments provided herein is acombination of an miRNA directed against an endogenous T cell receptorsubunit, wherein the expression of the miRNA is regulated by ariboswitch, which can be any of the riboswitches discussed herein.

In some embodiments, inhibitory RNA molecules can be provided onmultiple nucleic acid sequences that can be included on the same or adifferent transcriptional unit. For example, a first nucleic acidsequence can encode one or more inhibitory RNA molecules and beexpressed from a first promoter and a second nucleic acid sequence canencode one or more inhibitory RNA molecules and be expressed from asecond promoter. In illustrative embodiments, two or more inhibitory RNAmolecules are located on a first nucleic acid sequence that is expressedfrom a single promoter. The promoter used to express such miRNAs, aretypically promoters that are inactive in a packaging cell used toexpress a retroviral particle that will deliver the miRNAs in its genometo a target T cell, but such promoter is active, either constitutivelyor in an inducible manner, within a T cell. The promoter can be a Pol I,Pol II, or Pol III promoter. In some illustrative embodiments, thepromoter is a Pol II promoter.

Characterization and Commercial Production Methods

The present disclosure provides various methods and compositions thatcan be used in as research reagents in scientific experimentation andfor commercial production. Such scientific experimentation can includemethods for characterization of lymphocytes, such as NK cells and inillustrative embodiments, T cells using methods for geneticallymodifying, for example transducing lymphocytes provided herein. Suchmethods for example, can be used to study activation of lymphocytes andthe detailed molecular mechanisms by which activation makes such cellstransducible. Furthermore, provided herein are genetically modifiedlymphocytes that will have utility for example, as research tools tobetter understand factors that influence T cell proliferation andsurvival. Such genetically modified lymphocytes, such as NK cells and inillustrative embodiments T cells, can furthermore be used for commercialproduction, for example for the production of certain factors, such asgrowth factors and immunomodulatory agents, that can be harvested andtested or used in the production of commercial products.

The scientific experiments and/or the characterization of lymphocytescan include any of the aspects, embodiments, or subembodiments providedherein useful for analyzing or comparing lymphocytes. In someembodiments, T cells and/or NK cells can be transduced with thereplication incompetent recombinant retroviral particles provided hereinthat include polynucleotides. In some embodiments, transduction of the Tcells and/or NK cells can include polynucleotides that includepolynucleotides encoding polypeptides of the present disclosure, forexample, CARs, lymphoproliferative elements, and/or activation elements.In some embodiments, the polynucleotides can include inhibitory RNAmolecules as discussed elsewhere herein. In some embodiments, thelymphoproliferative elements can be chimeric lymphoproliferativeelements.

Treatment Methods

The present disclosure provides various treatment methods using a CAR. ACAR of the present disclosure, when present in a T lymphocyte or an NKcell, can mediate cytotoxicity toward a target cell. A CAR of thepresent disclosure binds to an antigen present on a target cell, therebymediating killing of a target cell by a T lymphocyte or an NK cellgenetically modified to produce the CAR. The ASTR of the CAR binds to anantigen present on the surface of a target cell.

The present disclosure provides methods of killing, or inhibiting thegrowth of, a target cell, the method involving contacting a cytotoxicimmune effector cell (e.g., a cytotoxic T cell, or an NK cell) that isgenetically modified to produce a subject CAR, such that the Tlymphocyte or NK cell recognizes an antigen present on the surface of atarget cell, and mediates killing of the target cell.

The present disclosure provides a method of treating a disease ordisorder in an individual having the disease or disorder, the methodincluding: a. introducing an expression vector including apolynucleotide sequence encoding a CAR into peripheral blood cellsobtained from the subject to produce a genetically engineered cytotoxiccell; and b. administering the genetically engineered cytotoxic cell tothe subject.

In methods provided herein that include administering geneticallymodified T cells and/or NK cells into a subject, especially where thesubject has, or is suspected of having cancer, the methods can furtherinclude delivering an effective dose of an immune checkpoint inhibitorto the subject. This checkpoint inhibitor delivery can occur before,after, or at the same time as administering the genetically modified Tcells and/or NK cells. Immune checkpoint inhibitors are known andvarious compounds are approved and in clinical development. Check pointmolecules, many of which are the target of checkpoint inhibitorcompounds, include the following, with some checkpoint inhibitors noted:

CD27. This molecule supports antigen-specific expansion of naïve T cellsand is vital for the generation of T cell memory. Celldex Therapeuticsis working on CDX-1127, an agonistic anti-CD27 monoclonal antibody.

CD28. This molecule is constitutively expressed on almost all human CD4+T cells and on around half of all CD8 T cells. CD28 was the target ofthe TGN1412 ‘superagonist’.

CD40. This molecule, found on a variety of immune system cells includingantigen presenting cells has CD40L, otherwise known as CD154 andtransiently expressed on the surface of activated CD4+ T cells, as itsligand. An anti-CD40 agonist monoclonal antibody was licensed to Roche.

CD122. This molecule, which is the Interleukin-2 receptor beta sub-unit,is known to increase proliferation of CD8+ effector T cells. NektarTherapeutics has developed NKTR-214, a CD122-biased immune-stimulatorycytokine.

CD137. When this molecule, also called 4-1BB, is bound by CD137 ligand,the result is T-cell proliferation. Pieris Pharmaceuticals has developedan engineered lipocalin that is bi-specific for CD137 and HER2.

OX40. This molecule, also called CD134, has OX40L, or CD252, as itsligand. Like CD27, OX40 promotes the expansion of effector and memory Tcells, however it is also noted for its ability to suppress thedifferentiation and activity of T-regulatory cells, and also for itsregulation of cytokine production. AstraZeneca has three drugs indevelopment targeting OX40: MEDI0562 is a humanised OX40 agonist;MEDI6469, murine OX4 agonist; and MEDI6383, an OX40 agonist[18]

GITR, short for Glucocorticoid-Induced TNFR family Related gene, promptsT cell expansion, including Treg expansion. TG Therapeutics is workingon anti-GITR antibodies.

ICOS. This molecule, short for Inducible T-cell costimulator, and alsocalled CD278, is expressed on activated T cells. Jounce Therapeutics isdeveloping an ICOS agonist.

A2AR. The Adenosine A2A receptor is regarded as an important checkpointin cancer therapy because adenosine in the immune microenvironment,leading to the activation of the A2a receptor, is negative immunefeedback loop and the tumor microenvironment has relatively highconcentrations of adenosine.

B7-H3, also called CD276, was originally understood to be aco-stimulatory molecule but is now regarded as co-inhibitory.MacroGenics is working on MGA271 is an Fc-optimized monoclonal antibodythat targets B7-H3.

B7-H4, also called VTCN1, is expressed by tumor cells andtumor-associated macrophages and plays a role in tumour escape.

BTLA. This molecule, short for B and T Lymphocyte Attenuator and alsocalled CD272, has HVEM (Herpesvirus Entry Mediator) as its ligand.

CTLA-4, short for Cytotoxic T-Lymphocyte-Associated protein 4 and alsocalled CD152, is the target of Bristol-Myers Squibb's compound Yervoy.

IDO, short for Indoleamine 2,3-dioxygenase, is a tryptophan catabolicenzyme with immune-inhibitory properties. Another important molecule isTDO, tryptophan 2,3-dioxygenase. IDO. Newlink Genetics and Incyte haveidentified IDO pathway inhibitors.

KIR, short for Killer-cell Immunoglobulin-like Receptor, is a receptorfor MHC Class I molecules on Natural Killer cells. Bristol-Myers Squibbhas developed Lirilumab, a monoclonal antibody to KIR.

LAG3, short for Lymphocyte Activation Gene-3, works to suppress animmune response by action to Tregs as well as direct effects on CD8+ Tcells. Bristol-Myers Squibb has developed an anti-LAG3 monoclonalantibody called BMS-986016.

PD-1, short for Programmed Death 1 (PD-1) receptor, has two ligands,PD-L1 and PD-L2. This checkpoint is the target of Merck & Co.'s melanomadrug Keytruda,

TIM-3, short for T-cell Immunoglobulin domain and Mucin domain 3,expresses on activated human CD4+ T cells and regulates Th1 and Th17cytokines.

VISTA (protein), Short for V-domain Ig suppressor of T cell activation,VISTA is primarily expressed on hematopoietic cells.

Subjects Suitable for Treatment

A variety of subjects are suitable for treatment with the methods andcompositions presented herein. Suitable subjects include any individual,e.g., a human or non-human animal who has a disease or disorder, who hasbeen diagnosed with a disease or disorder, who is at risk for developinga disease or disorder, who has had a disease or disorder and is at riskfor recurrence of the disease or disorder, who has been treated with anagent for the disease or disorder and failed to respond to suchtreatment, or who has been treated with an agent for the disease ordisorder but relapsed after initial response to such treatment.

Subjects suitable for treatment with an immunomodulatory method includeindividuals who have an autoimmune disorder; individuals who are organor tissue transplant recipients; and the like; individuals who areimmunocompromised; and individuals who are infected with a pathogen.

Exemplary Embodiments

Provided in this Exemplary Embodiments section are exemplary aspects andembodiments provided herein and further discussed throughout thisspecification. For the sake of brevity and convenience, all of thedisclosed aspects and embodiments and all of the possible combinationsof the disclosed aspects and embodiments are not listed in this section.It will be understood that embodiments are provided that are specificembodiments for many aspects, as discussed in this entire disclosure. Itis intended in view of the full disclosure herein, that any individualembodiment recited below or in this full disclosure can be combined withany aspect recited below or in this full disclosure where it is anadditional element that can added to an aspect or because it is narrowerelement for an element already present in an aspect. Such combinationsare discussed more specification in other sections of this detaileddescription.

In one aspect, provided herein is a replication incompetent recombinantretroviral particle including a polynucleotide including:

-   -   A. one or more transcriptional units operatively linked to a        promoter active in T cells and/or NK cells, wherein the one or        more transcriptional units encode a chimeric antigen receptor        (CAR); and    -   B. a pseudotyping element and an activation element (e.g. an NK        cell activation element or in illustrative embodiments a T cell        activation element) on its surface, wherein the activation        element is not encoded by a polynucleotide in the replication        incompetent recombinant retroviral particle.

In another aspect, provided herein is a replication incompetentrecombinant retroviral particle, including a polynucleotide includingone or more transcriptional units operatively linked to a promoteractive in T cells and/or NK cells, wherein the one or moretranscriptional units encode a first polypeptide including a chimericantigen receptor (CAR) and a second polypeptide including alymphoproliferative element. Various embodiments for such particle, suchas, but not limited to, lymphoproliferative elements, activationelements, pseudotyping elements, control elements, CARs, and otherelements, are provided herein in this section and in this disclosure.

In another aspect, provided herein is a replication incompetentrecombinant retroviral particle, comprising a polynucleotide comprisingone or more transcriptional units operatively linked to a promoteractive in T cells and/or NK cells, wherein the one or moretranscriptional units encode a first polypeptide comprising a chimericantigen receptor (CAR) and a second polypeptide comprising a chimericlymphoproliferative element, for example a constitutively activechimeric lymphoproliferative element. In illustrative embodiments, thechimeric lymphoproliferative element does not comprise a cytokinetethered to its cognate receptor or tethered to a fragment of itscognate receptor. Various embodiments for such particle, such as, butnot limited to, lymphoproliferative elements, activation elements,pseudotyping elements, control elements, CARs, and other elements, areprovided herein in this section and in this disclosure

In another aspect, provided herein is a replication incompetentrecombinant retroviral particle, comprising:

-   -   A. a polynucleotide comprising one or more transcriptional units        operatively linked to a promoter active in T cells and/or NK        cells, wherein the one or more transcriptional units encode a        chimeric antigen receptor (CAR); and    -   B. a pseudotyping element and a T cell activation element on its        surface, wherein the T cell activation element is not encoded by        a polynucleotide in the replication incompetent recombinant        retroviral particle, and wherein the T cell activation element        is an anti-CD3 scFvFc antibody. Various embodiments for such        particle, such as, but not limited to, lymphoproliferative        elements, activation elements, pseudotyping elements, control        elements, CARs, and other elements, are provided herein in this        section and in this disclosure

In another aspect, provided herein are replication incompetentrecombinant retroviral particles, comprising:

-   -   A. one or more pseudotyping elements capable of binding to a T        cell and/or an NK cell and facilitating membrane fusion of the        replication incompetent recombinant retroviral particles        thereto;    -   B. a polynucleotide comprising one or more transcriptional units        operatively linked to a promoter active in T cells and/or NK        cells, wherein the one or more transcriptional units encode a        first engineered signaling polypeptide comprising a chimeric        antigen receptor comprising an antigen-specific targeting        region, a transmembrane domain, and an intracellular activating        domain, and a second engineered signaling polypeptide comprising        at least one lymphoproliferative element; wherein expression of        the first engineered signaling polypeptide and/or the second        engineered signaling polypeptide are regulated by a control        element; and    -   C. an activation element on its surface, wherein the activation        element is capable of binding to a T cell and/or NK cell and is        not encoded by a polynucleotide in the replication incompetent        recombinant retroviral particles. Various embodiments for such        particle, such as, but not limited to, lymphoproliferative        elements, activation elements, pseudotyping elements, control        elements, CARs, and other elements, are provided herein in this        section and in this disclosure.

In some aspects provided herein is a genetically modified lymphocyte,such as a T cell or NK cell made by transducing resting T cells and/orresting NK cells according to a method comprising contacting the restingT cells and/or resting NK cells ex vivo, with any of the replicationincompetent recombinant retroviral particles provided herein, whereinsaid contacting facilitates transduction of the resting T cells and/orNK cells by the replication incompetent recombinant retroviralparticles, thereby producing genetically modified T cells and/or NKcells. In one embodiment, the replication incompetent recombinantretroviral particles comprise a pseudotyping element on their surfaceand a membrane-bound T cell activation element on their surface. Variousembodiments for such a cell, such as, but not limited to, contactingtimes, lymphoproliferative elements, activation elements, pseudotypingelements, control elements, and other elements, are provided herein inthis section and in this disclosure.

In another aspect provided herein is the use of any replicationincompetent recombinant retroviral particle in the manufacture of a kitfor genetically modifying a T cell or NK cell, wherein the use of thekit includes the steps of any of the methods aspects and embodimentsprovided herein. For example, in another aspect, provided herein is theuse of a replication incompetent recombinant retroviral particle in themanufacture of a kit for genetically modifying a T cell or NK cell,wherein the use of the kit includes: contacting the T cell or NK cell exvivo with the replication incompetent recombinant retroviral particle,wherein the replication incompetent recombinant retroviral particleincludes a pseudotyping element on a surface and typically a T cellactivation element on the surface, wherein said contacting facilitatestransduction of the T cell or NK cell by the replication incompetentrecombinant retroviral particle, thereby producing a geneticallymodified T cell or NK cell. In some embodiments, the T cell activationelement can be membrane-bound. In illustrative embodiments, the T cellactivating element activates the T cell through the T cell receptorassociated receptor complex. In some embodiments, the contacting can beperformed for between 1, 2, 3, 4, 5, 6, 7, or 8 hours on the low end ofthe range and 4, 5, 6, 7, 8, 10, 12, 15, 18, 21, and 24 hours on thehigh end of the range, for example, between 1 and 12 hours or between 1and 5 hours. In this use aspect and other use aspects herein, thereplication incompetent recombinant retroviral particle for use in themanufacture of a kit can include any of the replication incompetentrecombinant retroviral particle aspects and embodiments provided herein.Various other embodiments for such methods, such as but not limited toother contacting times, lymphoproliferative elements, activationelements, pseudotyping elements, percenttransfected/genetically-modified cells, control elements and otherelements are provided herein.

Provided herein in another aspect, is a method of transducing and/orgenetically modifying a lymphocyte, typically a T cell and/or an NKcell, and typically a resting T cell and/or resting NK cell, thatincludes contacting the lymphocyte with a replication incompetentrecombinant retroviral particle, wherein the replication incompetentrecombinant retroviral particle typically comprises a pseudotypingelement on its surface, wherein said contacting (which can also beconsidered incubating under contacting conditions) facilitatestransduction of the resting T cell and/or NK cell by the replicationincompetent recombinant retroviral particle, thereby producing thegenetically modified T cell and/or NK cell. The pseudotyping element istypically capable of binding the resting T cell and/or NK cell andtypically facilitating membrane fusion on its own or in conjunction withother protein(s) of the replication incompetent recombinant retroviralparticles. In some embodiments of the method, the replicationincompetent recombinant retroviral particle comprises an activationelement, such as a T cell activation element that activates a T cellthrough T cell receptor associated complex. Such an activation elementcan be an anti-CD3 antibody, for example an anti-CD3 scFv or an antiCD3scFvFc. Various exemplary and illustrative contacting times are providedherein. Various other embodiments for such methods, such as, but notlimited to, contacting times, lymphoproliferative elements, activationelements, pseudotyping elements, percenttransfected/genetically-modified cells, control elements and otherelements are provided herein.

In another aspect, provided herein is a method for genetically modifyingand/or transducing a lymphocyte, comprising contacting the lymphocyte exvivo, with a replication incompetent recombinant retroviral particle,wherein the replication incompetent recombinant retroviral particlecomprises a pseudotyping element on its surface and a membrane-bound Tcell activation element on its surface, wherein said contactingfacilitates transduction of the lymphocyte by the replicationincompetent recombinant retroviral particles, thereby producing agenetically modified lymphocyte. In some embodiments, the membrane-boundT cell activation element is an anti-CD3 antibody, for example ananti-CD3 scFv or an antiCD3 scFvFc. Various other embodiments for suchmethods, such as, but not limited to, contacting times,lymphoproliferative elements, activation elements, pseudotypingelements, percent transfected/genetically-modified cells, controlelements and other elements are provided herein.

Provided in another aspect herein, is a method of transducing restinglymphocytes of a subject, comprising contacting resting T cells and/orresting NK cells of a subject ex vivo, with replication incompetentrecombinant retroviral particles, wherein the replication incompetentrecombinant retroviral particles comprise a pseudotyping element ontheir surface that is capable of binding a resting T cell and/or restingNK cell and facilitating membrane fusion of the replication incompetentrecombinant retroviral particles thereto, wherein said contactingfacilitates transduction of the resting T cells and/or NK cells by thereplication incompetent recombinant retroviral particles, therebyproducing genetically modified T cells and/or NK cells. In illustrativeembodiments of this aspect, at least 10%, 20%, or 25% of the resting Tcells and/or NK cells, or between 10% and 70%, between 10% and 50%, orbetween 20% and 50% of T cells and/or NK cells are transduced as aresult of the process. Various other embodiments for such methods, suchas, but not limited to, contacting times, lymphoproliferative elements,activation elements, pseudotyping elements, percenttransfected/genetically-modified cells, control elements and otherelements are provided herein.

Provided in another aspect herein is a method for transducing resting Tcells and/or resting NK cells from isolated blood, comprising:

-   -   A. collecting blood from a subject;    -   B. isolating peripheral blood mononuclear cells (PBMCs)        comprising resting T cells and/or resting NK cells;    -   C. contacting the resting T cells and/or resting NK cells of the        subject ex vivo, with replication incompetent recombinant        retroviral particles, wherein the replication incompetent        recombinant retroviral particles comprise a pseudotyping element        on their surface that is capable of binding a resting T cell        and/or resting NK cell and facilitating membrane fusion of the        replication incompetent recombinant retroviral particles        thereto, wherein said contacting facilitates transduction of at        least 5% of the resting T cells and/or resting NK cells by the        replication incompetent recombinant retroviral particles,        thereby producing genetically modified T cells and/or NK cells,        thereby transducing resting T cells and/or NK cells. Various        other embodiments for such methods, such as, but not limited to,        contacting times, lymphoproliferative elements, activation        elements, pseudotyping elements, percent        transfected/genetically-modified cells, control elements and        other elements are provided herein.

Typically, a packaging cell line is used to produce the replicationincompetent recombinant retroviral particle as discussed elsewhereherein. In some embodiments, the packaging cell line can be a suspensioncell line. In illustrative embodiments, the packaging cell line can begrown in serum free media. In some embodiments, the lymphocyte can befrom a subject. In illustrative embodiments, the lymphocyte can be fromblood of a subject. Various other embodiments for such methods, such as,but not limited to, contacting times, lymphoproliferative elements,activation elements, pseudotyping elements, percenttransfected/genetically-modified cells, control elements and otherelements are provided herein.

In another aspect, provided herein is method of transducing a lymphocytewith a replication incompetent recombinant retroviral particlecomprising:

-   -   A. transfecting a packaging cell with a group of vectors        comprising components of the replication incompetent recombinant        retroviral particle, wherein the packaging cell is grown in        suspension in a serum-free media;    -   B. harvesting the replication incompetent recombinant retroviral        particle from the serum-free media; and    -   C. contacting the lymphocyte with the replication incompetent        recombinant retroviral particle. Various embodiments for such        methods, such as, but not limited to, contacting times,        lymphoproliferative elements, activation elements, pseudotyping        elements, percent transfected/genetically-modified cells,        control elements and other elements are provided herein.

In another aspect, provided herein is a method of transducing alymphocyte with a replication incompetent recombinant retroviralparticle comprising:

-   -   A. culturing a packaging cell in suspension in serum-free media,        wherein the packaging cell comprises nucleic acid sequences        encoding a packagable RNA genome of the replication incompetent        retroviral particle, a REV protein, a gag polypeptide, a pol        polypeptide, and a pseudotyping element;    -   B. harvesting the replication incompetent recombinant retroviral        particle from the serum-free media; and    -   C. contacting the lymphocyte with the replication incompetent        recombinant retroviral particle,        wherein the contacting is performed for less than 12 hours,        thereby transducing the lymphocyte. Various other embodiments        for such methods, such as, but not limited to, other contacting        times, lymphoproliferative elements, activation elements,        pseudotyping elements, percent transfected/genetically-modified        cells, control elements and other elements are provided herein.

Unless incompatible with, or already stated in an aspect, inillustrative embodiments, the packaging cell is an immortalized cellcomprising DNA nucleic acids stably integrated therein that encodes thepackagable RNA genome. In some embodiments, the gag polypeptide and thepol polypeptide are expressed from one or more inducible promoters andwherein the method further comprises during the culturing, adding atransactivator to induce expression of the gag polypeptide and the polpolypeptide from the one or more inducible promoters. Variousembodiments for such methods, such as, but not limited to, contactingtimes, lymphoproliferative elements, activation elements, controlelements, and other elements are provided herein.

Unless incompatible with, or already stated in an aspect, inillustrative embodiments of any of the methods aspects for geneticallymodifying and/or transducing lymphocytes, for example T lymphocytes, forgenetically modifying and expanding lymphocytes, for example Tlymphocytes, or for performing cellular therapy herein, or similarmethods the contacting can be carried out for between 15, 30 or 45minutes or 1, 2, 3, 4, 5, 6, 7, or 8 hours on the low end of the range,and between 2, 4, 6, 8, 10, 12, 18, 24, 36, 48, and 72 hours on the highend of the range. For example, in illustrative embodiments, thecontacting is carried out for between 2 and 24 hours, between 2 and 20hours, between 2 and 6 hours, between 1 and 20 hours, between 1 and 12hours, between 1 and 4 hours, between 4 and 12 hours, or between 4 and 8hours. In some embodiments, the contacting of a PBMC, NK cell and inillustrative embodiments a T cell with a replication incompetentrecombinant retroviral particle, in any of the methods, processes toproduce products, or uses thereof unless stated, or stated otherwise ina broadest aspect, provided herein, can be performed for between 1 and24 hours, for example, between 1 and 12 hours, between 1 and 6 hours. Insome embodiments, the contacting can be performed for less than 24hours, for example, less than 12 hours, less than 8 hours, or less than4 hours. In illustrative embodiments, the contacting is performed forbetween 1 and 14 hours, between 1 and 12 hours, between 1 and 6 hours,between 1 and 4 hours, or between 2 and 14 hours. Such methods can beperformed without prior activation.

Unless incompatible with, or already stated in an aspect, furtherembodiments of any of the above methods, use, or product by processaspects for transducing and/or genetically modifying a lymphocyte, forexample an NK cell or in illustrative embodiments, a T cell, can includeany of the embodiments of replication incompetent recombinant retroviralparticles, lymphoproliferative elements, CARs, pseudotyping elements,riboswitches, activation elements, membrane-bound cytokines, miRNAs,and/or other elements disclosed herein. In certain illustrativeembodiments, the retroviral particle is a lentiviral particle.

Unless incompatible with, or already stated in an aspect, in any of themethods, kits, uses, or compositions (e.g. polynucleotides, packingcells, or replication incompetent recombination retroviral particles)provided herein, the replication incompetent recombinant retroviralparticle(s) comprises or further comprises an activation element ontheir surface that is capable of activating a resting T cell and/or aresting NK cell. Typically, the activation element such as a T cellactivation element is not encoded by a polynucleotide in the replicationincompetent recombinant retroviral particle.

Unless incompatible with, or already stated in an aspect, in any of themethods, kits, uses, or compositions (e.g. polynucleotides, packingcells, or replication incompetent recombination retroviral particles)provided herein that recite a T cell and/or a NK cell, or a resting Tcell or a resting NK cell, in certain illustrative embodiments, the cellis a T cell.

Unless incompatible with, or already stated in an aspect, typically, therecombinant retroviral particle in any of the methods, kits, uses, orcompositions (e.g. polynucleotides, packing cells, or replicationincompetent recombination retroviral particles) provided herein, isreplication incompetent, i.e. cannot replicate. In illustrativeembodiments, the retrovirus is a lentivirus, such as a replicationincompetent or replication defective HIV lentivirus. In illustrativeembodiments, the retroviral particle is a lentiviral particle, such as areplication incompetent or replication defective HIV lentiviralparticle. Thus, in illustrative embodiments of any of the methods, kits,uses, or compositions (e.g. polynucleotides, packing cells, orreplication incompetent recombination retroviral particles) providedherein, if not already recited, or not already recited otherwise, thereplication incompetent recombinant retroviral particle is a lentiviralparticle and/or the genetically modified cell is a genetically modifiedT cell.

Unless incompatible with, or already stated in an aspect, inillustrative embodiments of any of the aspects provided herein, thelymphocytes can be T cells and/or NK cells. In further illustrativeembodiments, the T cells and/or NK cells can be resting T cells and/orresting NK cells.

Unless incompatible with, or already stated in an aspect, inillustrative embodiments of any of the methods aspects for transducing,genetically modifying and expanding lymphocytes or for performingadoptive cellular therapy herein, or similar methods, between 10% and75%, or 10% and 70%, or 10% and 60%, or 10% and 50%, or 10% and 25%, or20% and 75%, or 20% and 50%, or at least 10%, 20%, or 25% of resting Tcells are transduced and between 0% and 75% of NK cells are transduced.In other embodiments, between 5% and 80%, or 10% and 80%, or 10% and70%, or 10% and 60%, or 10% and 50%, or 10% and 25%, or 10% and 20%, or20% and 50% of resting NK cells are transduced.

Unless incompatible with, or already stated in an aspect, inillustrative embodiments of any of the methods aspects for geneticallymodifying and expanding lymphocytes or for performing adoptive cellulartherapy herein, or similar methods or any compositions provided herein,expression of said second engineered signaling polypeptide is regulatedby the control element.

Unless incompatible with, or already stated in an aspect, in any of themethod, use, product by process, or composition aspects herein thatinclude a genetically modified T cell or NK cell or a replicationincompetent recombinant retroviral particle, such genetically modified Tcell or NK cell or replication incompetent recombinant retroviralparticle can include a polynucleotide comprising one or moretranscriptional units operatively linked to a promoter active in T cellsand/or NK cells, wherein the one or more transcriptional units encode afirst polypeptide comprising a chimeric antigen receptor (CAR) and/or asecond polypeptide comprising a T cell lymphoproliferative element. Insome embodiments, expression of the CAR and/or the lymphoproliferativeelement are under the control of a control element or of differentcontrol elements for each. In some embodiments the CAR and thelymphoproliferative element can be expressed on the same polypeptide andin illustrative embodiments the CAR and the lymphoproliferative elementare expressed as separate polypeptides. In some embodiments the CAR isan MRB CAR. In some embodiments, the chimeric lymphoproliferativeelement is constitutively active, for example a constitutively activechimeric cytokine receptor. In such embodiments, the constitutivelyactive chimeric cytokine receptor can be under the control of a controlelement.

Unless incompatible with, or already stated in an aspect, in any of theaspects herein that include a polynucleotide comprising one or moretranscriptional units operatively linked to a promoter active in T cellsand/or NK cells, wherein the one or more transcriptional units encode afirst polypeptide comprising a chimeric antigen receptor (CAR) and/or asecond polypeptide comprising a T cell lymphoproliferative element, thepolynucleotide can further comprise one or more of a Kozak-relatedsequence, a WPRE element, and a multiple stop sequence, such as a doublestop codon or a triple stop codon, wherein one or more of the stopcodons of the double stop codon or the triple stop codon define atermination of a reading from of at least one of the one or moretranscriptional units. In certain embodiments, the polynucleotidecomprises a Kozak-type sequence selected from CCACCAT/UG(G),CCGCCAT/UG(G), GCCGCCGCCAT/UG(G), or GCCGCCACCAT/U(G). In certainembodiments both the −3 and +4 nucleotides relative to a start codon ofthe first nucleic acid sequence comprise a G. In another embodiment,which can be combined with the preceding embodiment that comprises aKozak-type sequence and/or the following embodiment that includes triplestop codon, the polynucleotide comprises a WPRE element. In someembodiments, the WPRE element is located 3′ of a stop codon of the oneor more transcriptional units and 5′ to a 3′ LTR of the polynucleotide.In another embodiment, which can be combined with either or both of thepreceding embodiments (i.e. an embodiment the polynucleotide comprises aKozak-type sequence and/or an embodiment wherein the polynucleotidecomprises a WPRE), the one or more transcriptional units terminates withone or more stop codons of a double stop codon or a triple stop codon.

Unless incompatible with, or already stated in an aspect, inillustrative embodiments of any of the methods, uses, and compositionsprovided herein that include a lymphoproliferative element, thelymphoproliferative element can be a chimeric lymphoproliferativeelement. In any of the embodiments herein that include a chimericlymphoproliferative element, the chimeric lymphoproliferative elementcan be a chimeric cytokine receptor. In any of the embodiments hereinthat include a chimeric lymphoproliferative element, the chimericlymphoproliferative element can be IL-7 tethered to IL-7 receptor alpha,or other than IL-7 tethered to IL-7 receptor alpha. In some embodiments,the chimeric cytokine receptor comprises IL-7 tethered to the IL-7receptor alpha, or a fragment thereof that retains the ability topromote proliferation of T cells and/or NK cells, and wherein thechimeric cytokine receptor is constitutively active. In someembodiments, the chimeric cytokine receptor comprises IL-7 covalentlyattached to a functional extracellular fragment of the IL-7 receptorcapable of binding to IL-7, an IL-7 transmembrane domain, and an IL-7signaling domain.

Unless incompatible with, or already stated in an aspect, in any of theaspects or embodiments herein that include a chimericlymphoproliferative element, the chimeric lymphoproliferative elementcan be other than a chimeric cytokine receptor. In illustrativeembodiments of any of the methods and compositions provided herein thatinclude a lymphoproliferative element, the chimeric lymphoproliferativeelement can be a chimeric cytokine receptor. In illustrative embodimentsof any of the methods and compositions provided herein that include achimeric cytokine receptor, the chimeric cytokine receptor comprises anintracellular signaling domain of an IL-7 receptor, an intracellularsignaling domain of an IL-12 receptor, an intracellular signaling domainof an IL-15/IL-2 receptor, such an IL-15/IL-2 beta receptor, anintracellular signaling domain of an IL-21 receptor, an intracellularsignaling domain of an IL-23 receptor, an intracellular signaling domainof an IL-27 receptor, an intracellular signaling domain of atransforming growth factor β (TGFβ) decoy receptor, or CD28. In someembodiments, the chimeric cytokine receptor comprises IL-7 fused to theIL-7 receptor.

Unless otherwise stated, or already stated in a broadest aspect, inillustrative embodiments of any of the methods and compositions providedherein that include a lymphoproliferative element, thelymphoproliferative element can comprise a T cell survival motif. The Tcell survival motif can comprise all or a functional fragment of IL-7receptor, IL-15 receptor, or CD28. In other embodiments, thelymphoproliferative element can include a cytokine or cytokine receptorpolypeptide, or a fragment thereof comprising a signaling domain. Forexample, the lymphoproliferative element can comprise an interleukinpolypeptide and the lymphoproliferative element can further comprise aportion of its cognate interleukin receptor polypeptide covalentlyattached via a linker. Typically, this portion of the cognateinterleukin receptor includes a functional portion of the extracellulardomain capable of binding the interleukin cytokine and the transmembranedomain. In some embodiments, the intracellular domain is anintracellular portion of the cognate interleukin receptor. In someembodiments, the intracellular domain is an intracellular portion of adifferent cytokine receptor that is capable of promoting lymphocyteproliferation and optionally survival ex vivo or in vitro in culture inthe absence of exposure of the lymphocyte to exogenous cytokines such asIL-15, IL-7, and in illustrative embodiments IL-2 and in the absence ofthe target for an ASTR of a CAR expressed by the lymphocytes duringculturing for 6, 7, 14, 21, or 35 days, or longer.

Unless incompatible with, or already stated in an aspect, in someembodiments the lymphoproliferative element is an interleukinpolypeptide covalently attached to its full length cognate interleukinreceptor polypeptide via a linker. Alternatively, thelymphoproliferative element can be an intracellular signaling domain ofan IL-7 receptor, an intracellular signaling domain of an IL-12receptor, an intracellular signaling domain of IL-23, an intracellularsignaling domain of IL-27, an intracellular signaling domain of an IL-15receptor, an intracellular signaling domain of an IL-21 receptor, or anintracellular signaling domain of a transforming growth factor β (TGFβ)decoy receptor.

Unless incompatible with, or already stated in an aspect, In someillustrative embodiments, the lymphoproliferative element isconstitutively active. Furthermore, the lymphoproliferative element caninclude a mutated IL-7 receptor or a fragment thereof, which can furtherinclude a constitutively active mutated IL-7 receptor or aconstitutively active fragment thereof. In some embodiments, thelymphoproliferative element or chimeric cytokine receptor comprises anIL7 InsPPCL mutant. Unless incompatible with, or already stated in anaspect, in any of the aspects herein that include a lymphoproliferativeelement, the lymphoproliferative element can be any of the CLEs recitedin the Lymphoproliferative Element section herein. For example, inillustrative embodiments the CLEs comprise domains from genesspecifically called out as especially noteworthy and/or Top Constructswith respect to the Examples herein.

In certain illustrative embodiments, the lymphoproliferative elementcomprises a cytokine receptor, or a fragment thereof that includes asignaling domain, that activates a Jak/STATS pathway. For example, suchlymphoproliferative element can include an intracellular domain ofIL21R, IL27R, IL31RA, LIFR, and OSMR. As shown in the experiments ofExamples 17 and 18 and Tables 7 to 17, chimeric lymphoproliferativeelements that include intracellular domains of these genes were foundamong the candidate chimeric polypeptides that induced the highestmagnitude of proliferation in PBMCs cultured in the absence of exogenouscytokines such as IL-2.

In some embodiments, the lymphoproliferative element can comprise anintracellular domain that is an interleukin or an interleukin receptorand that was a part of a Top Construct identified in Libraries 1A, 1.1A,2B, 2.1B, 3A, 3.1A, 3B, 3.1B, 4B, and/or 4.1B of Examples 17 and 18(Tables 7 to 17). In some embodiments, the lymphoproliferative elementcan comprise an intracellular domain that is a cytokine receptor andthat was a part of a Top Construct identified in Libraries 1A, 1.1A, 2B,2.1B, 3A, 3.1A, 3B, 3.1B, 4B, and/or 4.1B of Examples 17 and 18 (Tables7 to 17). In some embodiments, the lymphoproliferative element cancomprise an intracellular domain that includes at least one ITAM motifand that was a part of a Top Construct identified in Libraries 1A, 1.1A,2B, 2.1B, 3A, 3.1A, 3B, 3.1B, 4B, and/or 4.1B of Examples 17 and 18(Tables 7 to 17).

In illustrative embodiments, the lymphoproliferative element cancomprise an intracellular domain from IL7R, IL12RB1, IL15RA, or IL27RA,which were present in constructs that showed particularly noteworthyenrichments (i.e. elicited highest magnitude of proliferation) in aninitial screen and a repeated screen as detailed in Examples 17 and 18(Tables 19 to 23).

In illustrative embodiments, the lymphoproliferative element cancomprise an intracellular domain from the cytokine receptors CD27, CD40,CRLF2, CSF2RA, CSF3R, EPOR, FCER1G, FCGR2A, FCGR2C, GHR, IFNAR1, IFNAR2,IFNGR2, IL1R1, IL1RL1, IL2RA, IL2RG, IL3RA, IL5RA, IL6R, IL7R, IL9R,IL10RB, IL1IRA, IL12RB1, IL13RA1, IL13RA2, IL15RA, IL17RB, IL18R1,IL18RAP, IL20RB, IL22RA1, IL27RA, IL31RA, LEPR, MPL, OSMR, PRLR,TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, or TNFRSF18, which were present inconstructs that showed particularly noteworthy enrichments (i.e.elicited highest magnitude of proliferation) in an initial screen and arepeated screen as detailed in Examples 17 and 18 (Tables 19 to 23).

In illustrative embodiments, the lymphoproliferative element comprisesan intracellular domain from CD3D, CD3E, CD3G, CD79A, CD79B, FCER1G,FCGR2A, or FCGR2C, which include at least one ITAM motif and werepresent in constructs that showed particularly noteworthy enrichments(i.e. elicited highest magnitude of proliferation) in an initial screenand a repeated screen as detailed in Examples 17 and 18 (Tables 19 to23).

In some embodiments, the lymphoproliferative element in this paragraph,which were shown in examples 17 and 18 to be active in constructs withonly a single intracellular domain, can be the intracellular domain of alymphoproliferative element with either two or more intracellulardomains, or in illustrative embodiments a single intracellular domain,i.e. the lymphoproliferative element does not comprise two or moreintracellular domains. In illustrative embodiments, the intracellulardomain in a lymphoproliferative element comprises a domain from CD40,CRLF2, CSF2RA, CSF3R, EPOR, FCGR2A, IFNAR2, IFNGR2, IL1R1, IL3RA, IL7R,IL10RB, IL11RA, IL12RB1, IL13RA2, IL18RAP, IL31RA, MPL, MYD88, TNFRSF14,or TNFRSF18, which were present in constructs that showed particularlynoteworthy enrichments (i.e. elicited highest magnitude ofproliferation) in an initial screen and a repeated screen as detailed inExample 18 as single intracellular signaling domains (Tables 22 and 23).In illustrative embodiments, the intracellular domain in alymphoproliferative element comprises a domain from IL7R or IL12RB1,which were present in constructs that showed particularly noteworthyenrichments (i.e. elicited highest magnitude of proliferation) in aninitial screen and a repeated screen as detailed in Example 18 (Tables22 and 23). In some embodiments, the intracellular domain in alymphoproliferative element with a single intracellular domain can be acytokine receptor. In illustrative embodiments, the cytokine receptor ina lymphoproliferative element with a single intracellular domaincomprises a domain from CD40, CRLF2, CSF2RA, CSF3R, EPOR, FCGR2A,IFNAR2, IFNGR2, IL1R1, IL3RA, IL7R, IL10RB, IL11RA, IL12RB1, IL13RA2,IL18RAP, IL31RA, MPL, TNFRSF14, or TNFRSF18, which were present inconstructs that showed particularly noteworthy enrichments (i.e.elicited highest magnitude of proliferation) in an initial screen and arepeated screen as detailed in Example 18 (Tables 22 and 23). In someembodiments, the intracellular domain in a lymphoproliferative elementcan include at least one ITAM motif. In illustrative embodiments, theintracellular domain in a lymphoproliferative element that includes atleast one ITAM motif comprises a domain from FCGR2A, which was presentin constructs that showed particularly noteworthy enrichments (i.e.elicited highest magnitude of proliferation) in an initial screen and arepeated screen as detailed in Example 18 (Table 22).

In illustrative embodiments, the lymphoproliferative element cancomprise a costimulatory domain from CD27, CD28, OX40 (also referred toas TNFRSF4), GITR (also referred to as TNFRSF18), or HVEM (also referredto as TNFRSF14), which were present in constructs that showedparticularly noteworthy enrichments (i.e. elicited highest magnitude ofproliferation) in an initial screen and a repeated screen as detailed inExamples 17 and 18 (Tables 19 to 23).

In some embodiments, the lymphoproliferative element can be one of theconstructs M024-S190-S047, M025-S050-S197, M036-S170-S047,M012-S045-S048, M049-S194-S064, M025-S190-S050, M025-S190-S05,E013-T041-S186-S051, E013-T028-S186-S051, E014-T015-S186-S051,E011-T016-S186-S050, E011-T073-S186-S050, or E013-T011-S186-S211, all ofwhich stimulated proliferation of resting lymphocytes after transductionas shown in Example 21 (FIGS. 35 and 36 ). In certain embodiments,lymphoproliferative elements comprise an extracellular domain fromCSF3R, IL3RA, ICOS, CRLF, CSF2RA, LIFR, or CD40; a first intracellulardomain from MyD88, CD40, or MPL, and/or a second intracellular domainfrom CD27 or MyD88.

In some embodiments, the lymphoproliferative element can be theconstruct E013-T041-S186-S051, which stimulated proliferation of restinglymphocytes after transduction with a replication incompetentrecombinant retroviral particle displaying UCHT1scFvFc-GPI as shown andanalyzed in Example 22 (FIGS. 38A and B). In other embodiments thelymphoproliferative element is IL7-IL7RA-IL2RB, as shown and analyzed inExample 22.

As detailed in Example 17, for Libraries 1A and 1.1A, constructs withdomains from the cytokine receptors CD27, IL1RL1, IL6R, IL31RA, TNFRSF4,or TNFRSF18, showed particularly noteworthy enrichments (i.e. elicitedhighest magnitude of proliferation) in both Libraries 1A and 1.1A (Table19).

As detailed in Example 17, for Libraries 2B and 2.1B, constructs withdomains from the cytokine receptors CD40, FCER1G, FCGR2C, IFNGR2, GHR,IL10RB, IL11RA, IL13RA2, IL17RB, IL22RA1, TNFRSF14, or TNFRSF9, showedparticularly noteworthy enrichments (i.e. elicited highest magnitude ofproliferation) in both Libraries 2B and 2.1B (Table 20).

As detailed in Example 18, for Libraries 3A and 3.1A, constructs withdomains from the cytokine receptors CD27, CD40, CSF2RA, FCGR2A, MPL,OSMR, TNFRSF4, or TNFRSF18 showed particularly noteworthy enrichments(i.e. elicited highest magnitude of proliferation) in both Libraries 3Aand 3.1A (Table 21).

As detailed in Example 18, for Libraries 3B and 3.1B, constructs withdomains from the cytokine receptors CD27, CD40, CRLF2, CSF2RA, CSF3R,EPOR, FCER1G, FCGR2A, FCGR2C, IFNAR1, IFNAR2, IFNGR2, ILIRL1, IL2RG,IL3RA, IL5RA, IL6R, IL7R, IL9R, IL10RB, IL11RA, IL12RB1, IL13RA1,IL13RA2, IL15RA, IL18RAP, IL20RB, IL27RA, IL31RA, LEPR, MPL, OSMR, PRLR,TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, or TNFRSF18, showed particularlynoteworthy enrichments (i.e. elicited highest magnitude ofproliferation) in both Libraries 3B and 3.1B (Table 22).

As detailed in Example 18, for Libraries 4B and 4.1B, constructs withdomains from the cytokine receptors CD27, CD40, CRLF2, CSF2RA, CSF3R,EPOR, FCGR2A, FCGR2C, IFNAR2, IFNGR2, IL1R1, IL2RA, IL3RA, IL2RG, IL6R,IL7R, IL10RB, IL1IRA, IL31RA, IL13RA1, IL13RA2, IL18R1, MPL, OSMR,TNFRSF4, TNFRSF9, or TNFRSF18, showed particularly noteworthyenrichments (i.e. elicited highest magnitude of proliferation) in bothLibraries 4B and 4.1B (Table 23).

Unless incompatible with, or already stated in an aspect, in any of theaspects and embodiments disclosed herein such as method, use,compositions, and product by process aspects, that include alymphoproliferative element, in illustrative embodiments the geneticallymodified PBMCs, lymphocytes, or genetically modified T cells and/or NKcells are capable of promoting lymphocyte proliferation/expansion andoptionally survival ex vivo or in vitro in culture in the absence ofexposure of the cells to cytokines such as IL-15, IL-7, and inillustrative embodiments IL-2 and the target for an ASTR of a CARexpressed by the cells during culturing for 6, 7, 14, 21, or 35 days, orlonger. In any of the embodiments disclosed herein including a CAR and alymphoproliferative element, the genetically modified T cells and/or NKcells can be capable of engraftment in vivo in mice without exposure toa target recognized by the CAR.

Unless incompatible with, or already stated in an aspect, inillustrative embodiments of any of the methods and compositions providedherein that include a replication incompetent recombinant retroviralparticle(s), the replication incompetent recombinant retroviralparticles can comprise on their surface an activation element. Inillustrative embodiments, the activation element activates a T cellthrough T cell receptor associated complex. In some cases, theactivation element can activate a T cell alone. In other cases, anactivation element requires activation through the TCR receptor complexin order to further activate T cells. Accordingly, in some embodimentsthe activation element comprises:

-   -   A. a membrane-bound polypeptide capable of binding to CD3;        and/or    -   B. a membrane-bound polypeptide capable of binding to CD28.

Furthermore, the membrane-bound polypeptide capable of binding to CD3 isa polypeptide capable of binding to CD3 that can be fused to aheterologous GPI anchor attachment sequence and the membrane-boundpolypeptide capable of binding to CD28 can be a polypeptide capable ofbinding to CD28 that 8 is fused to a heterologous GPI anchor attachmentsequence. In some embodiments, the membrane-bound polypeptide capable ofbinding to CD28 is CD80, CD86, or a functional fragment thereof that iscapable of inducing CD28-mediated activation of Akt, such as theextracellular domain of CD80.

Unless incompatible with, or already stated in an aspect, inillustrative embodiments of any of the methods and compositions providedherein that are or include a replication incompetent recombinantretroviral particle, the activation element can be a membrane-boundpolypeptide capable of binding CD3 and/such as an anti-CD3 scFv or ananti-CD3 scFvFc. In illustrative embodiments of any of the methods andcompositions provided herein that include a replication incompetentrecombinant retroviral particle and an anti-CD3, the anti-CD3 can be ananti-CD3 scFvFc fused to a heterologous GPI anchor attachment sequence.In illustrative embodiments of any of the methods and compositionsprovided herein that include a replication incompetent recombinantretroviral particle, the membrane-bound polypeptide capable of bindingCD3 can be an anti-CD3 scFv bound to a CD14 GPI anchor attachmentsequence, and the membrane-bound polypeptide capable of binding to CD28can be CD80, or the extracellular domain thereof, bound to a CD16B GPIanchor attachment sequence. In illustrative embodiments of any of themethods and compositions provided herein that include a replicationincompetent recombinant retroviral particle, the replication incompetentrecombinant retroviral particles can comprise on their surface, ananti-CD3 scFv bound to a CD14 GPI anchor attachment sequence, CD80, orthe extracellular domain thereof, bound to a CD16B GPI anchor attachmentsequence, and a fusion polypeptide of IL-7, or an active fragmentthereof, and DAF comprising a GPI anchor attachment sequence. Inillustrative embodiments of any of the methods and compositions providedherein that include a replication incompetent recombinant retroviralparticle, the IL-7, or an active fragment thereof, and DAF fusion, theanti-CD3 scFV, and the CD80, or extracellular domain thereof eachcomprises a DAF signal sequence.

Unless otherwise stated, or already stated in a broadest aspect, inillustrative embodiments of any of the methods and compositions providedherein that are or include a replication incompetent recombinantretroviral particle(sthe replication incompetent recombinant retroviralparticles can comprise on their surface a membrane-bound cytokine. Themembrane-bound cytokine can be IL-7, IL-15, or an active fragmentthereof. In other embodiments, the membrane-bound cytokine is a fusionpolypeptide of IL-7, or an active fragment thereof, and DAF. Forexample, the fusion polypeptide can comprise the DAF signal sequence(nucleotides 1-34 of SEQ ID NO:286), IL-7 without its signal sequence(nucleotides 35-186 of SEQ ID NO:286), and a fragment of DAF thatincludes its GPI anchor attachment sequence (nucleotides 187-532 of SEQID NO:286).

Unless incompatible with, or already stated in an aspect, illustrativeembodiments of any of the method and composition aspects provided hereinthe pseudotyping element can comprise one or more heterologous envelopeproteins. In other examples, the pseudotyping element can include one ormore viral polypeptides recognized by T cells. The one or morepseudotyping elements can comprise a Measles Virus F polypeptide, aMeasles Virus H polypeptide, and/or a fragment thereof. The one or morepseudotyping elements can be cytoplasmic domain deletion variants of ameasles virus F polypeptide and/or a measles virus H polypeptide.

Unless incompatible with, or already stated in an aspect, inillustrative embodiments of any of the methods and compositions providedherein that include the control element is the control element canregulate the lymphoproliferative element, wherein thelymphoproliferative element is inactive or less active at promotingproliferation of the T cells and/or NK cells in the absence of thecompound, and wherein the compound is a molecular chaperone that bindsthe lymphoproliferative element and induces the activity of thelymphoproliferative element.

Unless incompatible with, or already stated in an aspect, inillustrative embodiments of any of the methods and compositions providedherein that include the control element, the control element can be apolynucleotide comprising a riboswitch. The riboswitch can be capable ofbinding a nucleoside analog and the compound that binds the controlelement is the nucleoside analog. The nucleoside analog can be anantiviral agent. The antiviral agent can be acyclovir or penciclovir.

Unless incompatible with, or already stated in an aspect, inillustrative embodiments of any of the methods and compositions providedherein that include an engineered signaling polypeptide, that includesan ASTR, the ASTR of either or both of the engineered signalingpolypeptides can bind to a tumor associated antigen. In someillustrative embodiments, the antigen-specific targeting region of thesecond engineered polypeptide is a microenvironment restrictedantigen-specific targeting region.

Unless incompatible with, or already stated in an aspect, inillustrative embodiments of any of the methods and compositions providedherein that include a replication incompetent recombinant retroviralparticle(s), if not explicitly recited in the broadest aspect, thereplication incompetent recombinant retroviral particles can encode arecognition domain for a monoclonal antibody approved biologic. In someembodiments, the recognition domain is expressed on the same transcriptas the chimeric antigen receptor and wherein the recognition domain isseparated from the chimeric antigen receptor by a ribosome skippingand/or cleavage signal. The ribosome skipping and/or cleavage signal canbe 2A-1. The recognition domain can include a polypeptide that isrecognized by an antibody that recognizes EGFR, or an epitope thereof.The recognition domain can be an EGFR mutant that is recognized by anEGFR antibody and expressed on the surface of transduced T cells and/orNK cells as another control mechanism provided herein. In relatedembodiments, the recognition domain can include a polypeptide that isrecognized by an antibody that recognizes EGFR, or an epitope thereof.

Unless incompatible with, or already stated in an aspect, any of themethod, use, product by process, the method can further includecollecting blood from a subject. The method can further include aftercontacting the resting T cells and/or resting NK cells of the subject exvivo, reintroducing the genetically modified T cells and/or NK cellsinto the subject. In certain embodiments, the resting T cells and/orresting NK cells that are contacted are from the blood of the subject,and wherein expansion of the genetically modified T cells and/or NKcells occurs in vivo within the subject. In certain embodiments, thesteps between the collecting blood and the reintroducing the geneticallymodified T cells and/or NK cells are performed in no more than 24 hours,18 hours, 12 hours, 8 hours, 6 hours, or 4 hours. Other times areprovided herein. In certain embodiments, the subject is not exposed to alymphodepleting agent within 7 days of performing the contacting, duringthe contacting, and/or within 7 days after the genetically modified Tcells and/or NK cells are reintroduced into the subject.

Provided herein in one aspect, is a method of transducing and/orgenetically modifying lymphocytes (e.g. T cell and/or NK cells), inillustrative embodiments resting lymphocytes (resting T cells and/or NKcells), of a subject, comprising contacting resting T cells and/orresting NK cells of a subject ex vivo, with replication incompetentrecombinant retroviral particles, wherein the replication incompetentrecombinant retroviral particles comprise a pseudotyping element ontheir surface and a membrane-bound anti-CD3 scFvFc antibody on theirsurface, that is capable of binding a resting T cell and/or resting NKcell and facilitating membrane fusion of the replication incompetentrecombinant retroviral particle thereto, wherein said contactingfacilitates transduction of the resting T cells and/or NK cells by thereplication incompetent recombinant retroviral particles, therebyproducing genetically modified T cells and/or NK cells.

Provided herein in one aspect, is a method for transducing and/orgenetically modifying resting T cells and/or resting NK cells fromisolated blood, comprising: collecting blood from a subject; isolatingperipheral blood mononuclear cells (PBMCs) comprising resting T cellsand/or resting NK cells; and contacting the resting T cells and/orresting NK cells of the subject ex vivo for an effective time, withreplication incompetent recombinant retroviral particles, wherein thereplication incompetent recombinant retroviral particle comprise apseudotyping element on their surface and a membrane-bound anti-CD3scFvFc antibody on their surface, thereby producing genetically modifiedT cells and/or NK cells, thereby transducing resting T cells and/or NKcells.

In the aspects herein for transducing and/or genetically modifying Tlymphocytes (e.g. T cell and/or NK cells) that include a membrane-boundanti-CD3 scFvFc antibody, the pseudotyping element in certainembodiments is the vesicular stomatitis virus envelope protein (VSV-G).In some embodiments, the replication incompetent retroviral particlesfurther comprise a membrane-bound polypeptide capable of binding toCD28, which can include, for example, an extracellular domain of CD80,CD86, or functional fragments thereof that retains the ability to bindCD28. In some embodiments, the anti-CD3 scFvFc antibody is fused to aheterologous GPI anchor attachment sequence. In some embodiments, theanti-CD3 scFvFc antibody is not encoded by a polynucleotide in thereplication incompetent recombinant retroviral particle.

In the aspects herein for transducing and/or genetically modifying Tlymphocytes (e.g. T cell and/or NK cells) that include a membrane-boundanti-CD3 scFvFc antibody, the recombinant retroviral particle canfurther include a polynucleotide comprising one or more transcriptionalunits operatively linked to a promoter active in T cells and/or NKcells, wherein the one or more transcriptional units encode a chimericantigen receptor. In some embodiments, the membrane-bound polypeptidecapable of binding to CD3 is not encoded by a polynucleotide in thereplication incompetent recombinant retroviral particle. In someembodiments, the anti-CD3 scFvFc antibody is not encoded by apolynucleotide in the replication incompetent recombinant retroviralparticle.

In another aspect provided herein is a method of transducing and/orgenetically modifying resting lymphocytes of a subject, comprisingcontacting resting T cells and/or resting NK cells of a subject ex vivo,with replication incompetent recombinant retroviral particles, whereinthe replication incompetent recombinant retroviral particles comprise apseudotyping element on their surface and a membrane-bound polypeptidecapable of binding to CD3 on their surface, but not a membrane-boundpolypeptide capable of binding to and activating CD28 on their surface,wherein said contacting facilitates transduction of the resting T cellsand/or NK cells by the replication incompetent recombinant retroviralparticles, thereby producing genetically modified T cells and/or NKcells.

In another aspect, provided herein is a method for transducing and/orgenetically modifying resting T cells and/or resting NK cells fromisolated blood, comprising: collecting blood from a subject; isolatingperipheral blood mononuclear cells (PBMCs) comprising resting T cellsand/or resting NK cells; and contacting the resting T cells and/orresting NK cells of the subject ex vivo for an effective time, withreplication incompetent recombinant retroviral particles, wherein thereplication incompetent recombinant retroviral particles comprise apseudotyping element on their surface and a membrane-bound polypeptidecapable of binding to CD3 on their surface, but not a membrane-boundpolypeptide capable of binding to and activating CD28 on their surface,thereby producing genetically modified T cells and/or NK cells, therebytransducing resting T cells and/or NK cells.

In the aspects herein for transducing and/or genetically modifyingresting T lymphocytes that include a membrane-bound polypeptide capableof binding to CD3 on their surface, but not a membrane-bound polypeptidecapable of binding to and activating CD28 on their surface, thepseudotyping element can be, for example, the vesicular stomatitis virusenvelope protein (VSV-G). In illustrative embodiments, themembrane-bound polypeptide capable of binding to CD3 is an anti-CD3scFvFc antibody, which in some embodiments is fused to a heterologousGPI anchor attachment sequence. In some embodiments of this aspect, thecontacting is performed for at least 2 hours, or between 2 hours and 24hours, or between 2 hours and 6 hours. In some embodiments, a detectablemarker is encoded by the genome of the replication incompetentrecombinant retroviral particle, and detected in the T cells and/or NKcells after the transduction. In some embodiments, the membrane-boundpolypeptide capable of binding to CD3 is not encoded by a polynucleotidein the replication incompetent recombinant retroviral particle. In someembodiments, a detectable marker is encoded by the genome of thereplication incompetent recombinant retroviral particles, and detectedin the T cells and/or NK cells after the transduction.

In another aspect, provided herein is a replication incompetentrecombinant retroviral particle, comprising: one or more pseudotypingelements; a polynucleotide comprising one or more transcriptional unitsoperatively linked to a promoter active in T cells and/or NK cells,wherein the one or more transcriptional units encode a chimeric antigenreceptor; and a pseudotyping element on its surface and an activationelement on its surface, wherein the activation element is capable ofbinding to a T cell and/or NK cell and is not encoded by apolynucleotide in the replication incompetent recombinant retroviralparticle, and wherein the activation element is an anti-CD3 scFvFcantibody.

In another aspect, provided herein is a replication incompetentrecombinant retroviral particle, comprising: one or more pseudotypingelements capable of binding to a T cell and/or an NK cell andfacilitating membrane fusion of the replication incompetent recombinantretroviral particle thereto;

a polynucleotide comprising one or more transcriptional unitsoperatively linked to a promoter active in T cells and/or NK cells,wherein the one or more transcriptional units encode a chimeric antigenreceptor; and a pseudotyping element on its surface and an activationelement on its surface, wherein the activation element is capable ofbinding to a T cell and/or NK cell and is not encoded by apolynucleotide in the replication incompetent recombinant retroviralparticle, and wherein the activation elements is a membrane-boundpolypeptide capable of binding to CD3 on their surface, but not amembrane-bound polypeptide capable of binding to and activating CD28 ontheir surface.

In some embodiments of the replication incompetent recombinantretroviral particle aspects herein, the recombinant retroviral particlefurther comprises a polynucleotide comprising one or moretranscriptional units operatively linked to a promoter active in T cellsand/or NK cells, wherein the one or more transcriptional units encode achimeric antigen receptor. In some embodiments in these aspects, themembrane-bound polypeptide capable of binding to CD3 is not encoded by apolynucleotide in the replication incompetent recombinant retroviralparticle. In some embodiments of these aspects, the anti-CD3 scFvFcantibody is not encoded by a polynucleotide in the replicationincompetent recombinant retroviral particle.

In any of the aspects and embodiments herein, such as those including apolypeptide or a nucleic acid encoding the same, that include anactivation element, for example present on the surface of replicationincompetent recombinant retroviral particles, the activation element canfurther include, for example as a fusion polypeptide with a polypeptidecapable of binding CD28 or in illustrative embodiments, CD3, adimerizing motif that can be active in the absence of a dimerizingagent. In some embodiments, the activation element can be an anti-CD3single chain antibody and the dimerizing motif can be selected from thegroup consisting of: a leucine zipper polypeptide, CD69, CD71, CD72,CD96, Cd105, Cd161, Cd162, Cd249, CD271, and Cd324, as well as mutantsand/or active fragments thereof that retain the ability to dimerize.

In any of the aspects and embodiments herein, such as those includingthat include a polypeptide or a nucleic acid encoding the same, thatinclude an activation element, for example present on the surface ofreplication incompetent recombinant retroviral particles, the activationelement can further include, for example as a fusion polypeptide with apolypeptide capable of binding CD28 or in illustrative embodiments, CD3,a dimerizing motif that can be active in the presence of a dimerizingagent. In some embodiments, the activation element can be an anti-CD3single chain antibody and the dimerizing motif can be selected from thegroup consisting of: FKBP and rapamycin or analogs thereof, GyrB andcoumermycin or analogs thereof, DHFR and methotrexate or analogsthereof, or DmrB and AP20187 or analogs thereof, as well as mutantsand/or active fragments of the recited dimerizing proteins that retainthe ability to dimerize.

In any of the aspects and embodiments herein, such as those includingthat include a polypeptide or a nucleic acid encoding the same, thatinclude an activation element, for example present on the surface ofreplication incompetent recombinant retroviral particles, the activationelement can further include, an antiCD3-scFvFc-GPI moiety (UCHT1) andoptionally a VSV-G.

In one embodiment of the above aspect said polynucleotide furthercomprises one or more of a Kozak-type sequence, a WPRE element, and adouble stop codon or a triple stop codon, wherein one or more of thestop codons of the double stop codon or the triple stop codon define atermination of a reading from of at least one of the one or moretranscriptional units. In certain embodiments, the polynucleotidecomprises a Kozak-type sequence selected from CCACCAT/UG(G),CCGCCAT/UG(G), GCCGCCGCCAT/UG(G), or GCCGCCACCAT/U(G). In certainembodiments both the −3 and +4 nucleotides relative to a start codon ofthe first nucleic acid sequence comprise a G. In another embodiment,which can be combined with the preceding embodiment that comprises aKozak-type sequence and/or the following embodiment that includes triplestop codon, the polynucleotide comprises a WPRE element. In someembodiments, the WPRE element is located 3′ of a stop codon of the oneor more transcriptional units and 5′ to a 3′ LTR of the polynucleotide.In another embodiment, which can be combined with either or both of thepreceding embodiments (i.e. an embodiment the polynucleotide comprises aKozak-type sequence and/or an embodiment wherein the polynucleotidecomprises a WPRE), the one or more transcriptional units terminates withone or more stop codons of a double stop codon or a triple stop codon.

In certain embodiments of any of the aspects provided herein thatinclude an ASTR, the ASTR can be an anti-tag ASTR. Such methods canfurther include, for example, a tag-conjugated antibody that binds to atarget molecule, such as a target protein on a target cell.

In any of the embodiments and aspects provided herein, that include a Bcell, T cell, or NK cell, the cell can be an allogeneic cell, or it canbe other than an allogeneic cell.

In any of the aspects and embodiments disclosed herein, the introductionof DNA into PBMCs, B cells, T cells and/or NK cells and optionally theincorporation of the DNA into the host cell genome, can be performedusing methods that do not utilize replication incompetent recombinantretroviral particles. For example, other viral vectors can be utilized,such as those derived from adenovirus, adeno-associated virus, or herpessimplex virus-1, as non-limiting examples. In other embodiments, theseaspects and embodiments can include transfecting and/or transducingtarget cells with non-viral vectors. In any of these embodimentsdisclosed herein that utilize non-viral vectors to transfect targetcells, the non-viral vectors, including naked DNA, can be introducedinto the target cells, such as for example, PBMCs, B cells, T cellsand/or NK cells using methods that include electroporation,nucleofection, liposomal formulations, lipids, dendrimers, cationicpolymers such as poly(ethylenimine) (PEI) and poly(l-lysine) (PLL),nanoparticles, cell-penetrating peptides, microinjection, and/ornon-integrating lentiviral vectors. In some embodiments of methods andcompositions provided herein, DNA can be integrated into the genomeusing transposon-based carrier systems by co-transfection,co-nucleofection or co-electroporation of target DNA as plasmidcontaining the transposon ITR fragments in 5′ and 3′ ends of the gene ofinterest and transposase carrier system. A transposase or other enzyme,for example CRISPR or TALEN, used in these methods can be co-transfectedwith target plasmid as DNA, mRNA or protein.

In another aspect, provided herein are replication incompetentrecombinant retroviral particles, each comprising:

-   -   A. a pseudotyping element on its surface that is capable of        binding to a T cell and/or NK cell and facilitating membrane        fusion of the replication incompetent recombinant retroviral        particle thereto, wherein said pseudotyping element comprises        cytoplasmic domain deletion variants of a measles virus F        polypeptide and/or a measles virus H polypeptide;    -   B. a polynucleotide comprising one or more transcriptional units        operatively linked to a promoter active in T cells and/or NK        cells, wherein the one or more transcriptional units encode a        first engineered signaling polypeptide comprising a chimeric        antigen receptor comprising an antigen-specific targeting        region, a transmembrane domain, and an intracellular activating        domain, and a second engineered signaling polypeptide comprising        a constitutively active IL-7 receptor mutant; wherein expression        of the IL-7 receptor mutant is regulated by a riboswitch that        binds a nucleoside analog antiviral drug; and    -   C. a polypeptide capable of binding to CD3 and a polypeptide        capable of binding to CD28, wherein said polypeptides are        expressed on the surface of a replication incompetent        recombinant retroviral particle; are capable of binding to a T        cell and/or NK cell; and are not encoded by a polynucleotide in        the replication incompetent recombinant retroviral particle. In        illustrative embodiments of this aspect, binding of the        nucleoside analog antiviral drug to the riboswitch increases        expression of the IL-7 receptor mutant.

In one aspect, provided herein is a method for genetically modifying andexpanding lymphocytes of a subject, comprising:

-   -   A. contacting resting T cells and/or NK cells of the subject ex        vivo without requiring prior ex vivo stimulation, with        replication incompetent recombinant retroviral particles        comprising:        -   i. a pseudotyping element on its surface that is capable of            binding to a T cell and/or NK cell and facilitating membrane            fusion of the replication incompetent recombinant retroviral            particles thereto; and        -   ii. a polynucleotide comprising one or more transcriptional            units operatively linked to a promoter active in T cells            and/or NK cells, wherein the one or more transcriptional            units encode a first engineered signaling polypeptide            regulated by a control element, wherein said first            engineered signaling polypeptide comprises at least one            lymphoproliferative element, wherein said contacting            facilitates transduction of at least some of the resting T            cells and/or NK cells by the replication incompetent            recombinant retroviral particles, thereby producing            genetically modified T cells and/or NK cells;    -   B. introducing the genetically modified T cells and/or NK cells        into the subject; and    -   C. exposing the genetically modified T cells and/or NK cells in        vivo to a compound that binds the control element to affect        expression of the first engineered signaling polypeptide and        promote and/or potentiate expansion, engraftment, and/or        persistence of the lymphocytes in vivo, thereby genetically        modifying and expanding lymphocytes of the subject. In        illustrative embodiments, the transduction is carried out        without ex vivo stimulation.

In the above aspect and any of the method aspects for geneticallymodifying and expanding lymphocytes or for performing cellular therapyherein, if not recited in or incompatible with the aspect, in certainembodiments the polynucleotide further comprises a transcriptional unitthat encodes a second engineered signaling polypeptide comprising afirst chimeric antigen receptor comprising an antigen-specific targetingregion (ASTR), a transmembrane domain, and an intracellular activatingdomain.

In another aspect, provided herein is a method for performing adoptivecell therapy on a subject, comprising:

-   -   A. collecting blood from the subject;    -   B. contacting resting T cells and/or NK cells from the blood of        the subject ex vivo with replication incompetent recombinant        retroviral particles, wherein the replication incompetent        recombinant retroviral particles comprise        -   i. a pseudotyping element on their surface that is capable            of binding to a T cell and/or NK cell and facilitating            membrane fusion of the replication incompetent recombinant            retroviral particles thereto; and        -   ii. a polynucleotide comprising one or more transcriptional            units operatively linked to a promoter active in T cells            and/or NK cells, wherein the one or more transcriptional            units encode a first engineered signaling polypeptide            comprising at least one lymphoproliferative element whose            expression is regulated by a control element, and a second            engineered signaling polypeptide comprising a chimeric            antigen receptor comprising an antigen-specific targeting            region (ASTR), a transmembrane domain, and an intracellular            activating domain,        -   wherein said contacting results in at least some of the            resting T cells and/or NK cells becoming genetically            modified; and    -   C. reintroducing the genetically modified T cells and/or NK        cells into the subject, wherein expansion, engraftment, and/or        persistence of the genetically modified T cells and/or NK cells        occurs in vivo within the subject, and wherein the method        between the collecting blood and the reintroducing the        genetically modified T cells and/or NK cells is performed in no        more than 24 hours, thereby performing adoptive cell therapy on        the subject.

Provided in another aspect herein is a method for performing adoptivecell therapy on a subject, comprising:

-   -   A. collecting blood from a subject;    -   B. isolating peripheral blood mononuclear cells (PBMCs)        comprising resting T cells and/or resting NK cells;    -   C. contacting the resting T cells and/or resting NK cells of the        subject ex vivo, with replication incompetent recombinant        retroviral particles, wherein the replication incompetent        recombinant retroviral particles comprise a pseudotyping element        on their surface that is capable of binding a resting T cell        and/or NK cell and facilitating membrane fusion of the        replication incompetent recombinant retroviral particles        thereto, wherein said contacting facilitates transduction of the        resting T cells and/or NK cells by the replication incompetent        recombinant retroviral particles, thereby producing genetically        modified T cells and/or NK cells; and    -   D. reintroducing the genetically modified cells into the subject        within 24 hours of collecting blood from the subject, thereby        performing adoptive cell therapy in the subject.

In illustrative embodiments of any of the methods aspects forgenetically modifying and expanding lymphocytes or for performingadoptive cellular therapy herein, or similar methods, if not explicitlyrecited in the broadest aspect the method can further comprise exposingthe genetically modified T cells and/or NK cells in vivo to a compoundthat binds the control element to affect expression of the firstengineered signaling polypeptide and optionally the second engineeredsignaling polypeptide, and to promote expansion, engraftment, and/orpersistence of the lymphocytes in vivo.

In illustrative embodiments of any of the methods aspects forgenetically modifying and expanding lymphocytes or for performingadoptive cellular therapy herein, or similar methods, if not explicitlyrecited in the broadest aspect, the genetically modified T cells and/orNK cells undergo 8, 7, 6, 5, 4, 3 or fewer cell divisions ex vivo priorto being introduced or reintroduced into the subject.

In illustrative embodiments of any of the methods aspects forgenetically modifying and expanding lymphocytes or for performingcellular therapy herein, or similar methods, if not explicitly recitedin the broadest aspect, expansion, engraftment, and/or persistence ofgenetically modified T cells and/or NK cells in vivo is dependent oneither the presence or absence of the compound that binds the controlelement, and in illustrative embodiments, is dependent on the presenceof the compound that binds the control element.

In illustrative embodiments of any of the methods aspects forgenetically modifying and expanding lymphocytes or for performingadoptive cellular therapy herein, or similar methods, if not explicitlyrecited in the broadest aspect, the subject is not exposed to alymphodepleting agent within 7, 14, or 21 days of performing thecontacting, during the contacting, and/or within 7, 14, or 21 days afterthe modified T cells and/or NK cells are introduced into the subject. Inother embodiments, the subject is not exposed to a lymphodepleting agentduring the contacting.

In illustrative embodiments of any of the methods aspects forgenetically modifying and expanding lymphocytes or for performingcellular therapy herein, or similar methods, if not explicitly recitedin the broadest aspect, the resting T cells and/or resting NK cells arein contact with the replication incompetent recombinant retroviralparticles for between 15 minutes and 12 hours.

In illustrative embodiments of any of the methods aspects forgenetically modifying and expanding lymphocytes or for performingadoptive cellular therapy herein, or similar methods, if not explicitlyrecited in the broadest aspect, the method further includes the step ofseparating the replication incompetent recombinant retroviral particlesfrom the T cells and/or NK cells after the contacting. In methods thatinclude an introducing or reintroducing, but the separating is performedbefore the introducing. In illustrative embodiments of any of themethods aspects for genetically modifying and expanding lymphocytes orfor performing cellular therapy herein, or similar methods, if notexplicitly recited in the broadest aspect, said exposing step comprisesadministering a dose of the compound to the subject prior to or duringthe contacting, and/or after the genetically modified T cells and/or NKcells have been introduced into the subject.

In illustrative embodiments of any of the methods aspects forgenetically modifying and expanding lymphocytes or for performingadoptive cellular therapy herein, or similar methods, if not explicitlyrecited in the broadest aspect, the method comprises collecting bloodcomprising the T cells and/or the NK cells from the subject prior tocontacting the T cells and/or NK cells ex vivo with the replicationincompetent recombinant retroviral particles, and wherein theintroducing is reintroducing. For example, between 20 and 250 ml ofblood are withdrawn from the subject.

In illustrative embodiments of any of the methods aspects forgenetically modifying and expanding lymphocytes or for performingcellular therapy herein, or similar methods, if not explicitly recitedin the broadest aspect, no more than 8, 12, 24, or 48 hours pass betweenthe time blood is collected from the subject and the time the modified Tcells and/or NK cells are reintroduced into the subject.

In illustrative embodiments of any of the methods aspects forgenetically modifying and expanding lymphocytes or for performingcellular therapy herein, or similar methods, if not explicitly recitedin the broadest aspect, between 4 or 8 hours on the low end and 12. 24,36, or 48 hours on the high end of the range pass between the time bloodis collected from the subject and the time the modified T cells and/orNK cells are reintroduced into the subject.

In aspects and embodiments provided herein that include administeringgenetically modified T cells and/or NK cells into a subject, especiallywhere the subject has, or is suspected of having cancer, the methods canfurther include delivering an effective amount of an immune checkpointinhibitor to the subject.

In illustrative embodiments of any of the methods aspects forgenetically modifying and expanding lymphocytes or for performingadoptive cellular therapy herein, or similar methods, if not explicitlyrecited in the broadest aspect, all steps after the blood is collectedand before the blood is reintroduced, are performed in a closed systemin which a person monitors the closed system throughout the processing.In another embodiment, after the blood is collected and before the bloodis reintroduced, are performed in a closed system that remains in thesame room with the subject.

In illustrative embodiments of any of the methods and compositionsprovided herein that include one or more transcriptional units or one ormore engineered signaling polypeptides, if not recited in the broadestaspect, one of the transcriptional units or one of the engineeredsignaling polypeptides can encode or comprise or further comprise anantigen-specific targeting region (ASTR) and a transmembrane domainconnecting the ASTR to the lymphoproliferative element. The ASTR of thisengineered signaling polypeptide is capable of binding to a first tumorantigen and where present, the ASTR of the second engineered signalingpolypeptide is capable of binding to a second tumor antigen. Inillustrative embodiments, the first engineered signaling polypeptideand/or the second engineered signaling polypeptide further comprise aco-stimulatory domain. Furthermore, the first engineered signalingpolypeptide and/or the second engineered signaling polypeptide furthercomprise a stalk. Furthermore, the first engineered signalingpolypeptide further comprises an intracellular activating domain. Theintracellular activating domain on the first engineered signalingpolypeptide and/or the second engineered signaling polypeptide can bederived from CD3 zeta. In illustrative embodiments of any of the methodsand compositions provided herein that include a lymphoproliferativeelement, the lymphoproliferative element can comprise MPL or can be MPL,or a variant and/or fragment thereof, including a variant and/orfragment that includes at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or100% of the intracellular domain of MPL, with or without a transmembraneand/or extracellular domain of MPL, and/or has at least 75, 80, 85, 90,95, 96, 97, 98, 99, or 100% sequence identity to the intracellulardomain of MPL, with or without a transmembrane and/or extracellulardomain of MPL, wherein the variant and/or fragment retains the abilityto promote cell proliferation of PBMCs, and in some embodiments T cells.

In any of the methods or compositions provided herein that include alymphoproliferative element, the lymphoproliferative element can includean inhibitory RNA molecule, such as, e.g., a miRNA or shRNA, thatstimulates the STATS pathway or inhibits the SOCS pathway. For example,an inhibitory RNA molecule can bind to a nucleic acid encoding a proteinselected from the group consisting of: ABCG1, SOCS, TGFbR2, SMAD2, cCBL,and PD1. In illustrative embodiments for any of the replicationincompetent recombinant retroviral particles or transduced cellsprovided herein, or methods including the same, such replicationincompetent recombinant retroviral particles or transduced cells canencode two or more inhibitory RNA molecules, such as, e.g., a miRNA orshRNA, within an intron, in some embodiments, 1, 2, 3, or 4 inhibitoryRNA molecules that bind nucleic acids encoding one or more of thefollowing target endogenous T cell expressed genes: PD-1; CTLA4; TCRalpha; TCR beta; CD3 zeta; SOCS; SMAD2; miR-155; IFN gamma; cCBL;TRAIL2; PP2A; or ABCG1. For example, in one embodiment, a combination ofmiRNAs targeting any of the following can be included in a genome of areplication incompetent recombinant retroviral particle or transducedcell: TCR alpha, CD3 zeta, IFN gamma, and PD-1; and in anotherembodiment SOCS 1, IFN gamma, TCR alpha, and CD3 zeta.

In illustrative embodiments of any of the methods and compositionsprovided herein, the replication incompetent recombinant retroviralparticles, mammalian cells, and/or packaging cells, can comprise a Vpupolypeptide. The Vpu polypeptide can be, for example, a fusionpolypeptide, and in some examples, especially in packaging cells, amembrane bound Vpu polypeptide. In illustrative embodiments of any ofthe methods and compositions provided herein, the replicationincompetent recombinant retroviral particles, mammalian cells, and/orpackaging cells, can comprise both a Vpu polypeptide and a Vpxpolypeptide.

In illustrative embodiments of any of the methods and compositionsprovided herein, the replication incompetent recombinant retroviralparticles, mammalian cells, and/or packaging cells, can comprise a Vpxpolypeptide. The Vpx polypeptide can be, for example, a fusionpolypeptide, and in some examples, especially in packaging cells, amembrane bound Vpx polypeptide.

In any of the methods or compositions provided herein, the one or morepseudotyping elements can include a vesicular stomatitis virus envelopeprotein (VSV-G), a feline endogenous virus (RD114) envelope protein, anoncoretroviral amphotropic envelope protein, or an oncoretroviralecotropic envelope protein, or functional fragments thereof.

In one aspect, provided herein is a genetically modified T cell or NKcell, wherein the T cell or NK cell has been genetically modified toexpress a first engineered signaling polypeptide comprising alymphoproliferative element and a second engineered signalingpolypeptide comprising a CAR that includes an antigen-specific targetingregion (ASTR), a transmembrane domain, and an intracellular activatingdomain.

In another aspect, provided herein is a genetically modified T cell orNK cell comprising a pseudotyping element on a surface of the T cell orNK cell and an activation element on the surface of the T cell or NKcell, wherein the T cell or NK cell has been genetically modified toexpress a first engineered signaling polypeptide comprising alymphoproliferative element and a second engineered signalingpolypeptide comprising a chimeric antigen receptor that includes anantigen-specific targeting region (ASTR), a transmembrane domain, and anintracellular activating domain.

Provided herein in another aspect is a genetically modified T celland/or NK cell comprising:

-   -   A. a first engineered signaling polypeptide comprising at least        one lymphoproliferative element; and    -   B. a second engineered signaling polypeptide comprising a        chimeric antigen receptor comprising an antigen-specific        targeting region (ASTR), a transmembrane domain, and an        intracellular activating domain.

In some embodiments, the genetically modified T cells and/or NK cellsare capable of survival in ex vivo culture in the absence of IL-2. Insome embodiments, the genetically modified T cells and/or NK cellscomprise a chimeric cytokine receptor. In some embodiments, thegenetically modified T cells and/or NK cells do not comprise a chimericcytokine receptor.

In any of the methods provided herein that include a mammalian packagingcell, including a replication incompetent recombinant retroviralparticle packaging system aspect, or a method for making a replicationincompetent recombinant retroviral particle, for example, thepackageable RNA genome is encoded by a polynucleotide operably linked toa promoter, wherein said promoter is either constitutively active orinducible by either the first transactivator or the secondtransactivator. The packageable RNA genome can be encoded by apolynucleotide operably linked to a promoter, wherein said promoter isinducible by the second transactivator. A promoter used herein to driveexpression of the first and/or second engineered signaling polypeptide,is typically active in target cells, for example lymphocytes, PBLs,T-cells and/or NK cells, but in illustrative embodiments, is not activein the packaging cell line. The second transactivator can regulate theexpression of an activation element capable of binding to and activatingthe target cell. In any of the methods provided herein that include amammalian packaging cell, including a replication incompetentrecombinant retroviral particle packaging system aspect, or a method formaking a replication incompetent recombinant retroviral particle, forexample, the packageable RNA genome in some embodiments, expression ofthe packageable RNA genome can be regulated by the secondtransactivator.

Furthermore, the packageable RNA genome can comprise, from 5′ to 3′:

-   -   1.) a 5′ long terminal repeat, or active fragment thereof;    -   2.) a nucleic acid sequence encoding a retroviral cis-acting RNA        packaging element;    -   3.) a nucleic acid sequence encoding a first target polypeptide        and/or a nucleic acid sequence encoding one or more (e.g. two or        more) inhibitory RNA molecules;    -   4.) a promoter that is active in the target cell; and    -   5.) a 3′ long terminal repeat, or active fragment thereof.

In some embodiments, the nucleic acid sequence encoding the first targetpolypeptide is in reverse orientation to an RNA encoding retroviralcomponents for packaging and assembly and the 5′ LTR.

In any of the methods provided herein that include a mammalian packagingcell, including a replication incompetent recombinant retroviralparticle packaging system aspect, or a method for making a replicationincompetent recombinant retroviral particle, for example, the firsttarget polypeptide comprises a first engineered signaling polypeptideand wherein said first engineered signaling polypeptide comprises atleast one lymphoproliferative element. The packageable RNA genome canfurther comprises a nucleic acid sequence encoding a second targetpolypeptide. The second target polypeptide can comprise a secondengineered signaling polypeptide including a chimeric antigen receptorcomprising:

-   -   1.) a first antigen-specific targeting region;    -   2.) a first transmembrane domain; and    -   3.) a first intracellular activating domain.

In any of the methods provided herein that include a mammalian packagingcell, including a replication incompetent recombinant retroviralparticle packaging system aspect, or a method for making a replicationincompetent recombinant retroviral particle, for example, the mammaliancell, for example the packaging cell can include a nucleic acid sequenceencoding Vpu polypeptide, for example on the second or an optional thirdtranscriptional unit, or on an additional transcriptional unit that isoperably linked to the first inducible promoter. In any of the methodsprovided herein that include a mammalian packaging cell, including areplication incompetent recombinant retroviral particle packaging systemaspect, or a method for making a replication incompetent recombinantretroviral particle, for example, the mammalian cell, for example thepackaging cell can include a nucleic acid sequence encoding both a Vpxpolypeptide and a Vpu polypeptide, for example on the second or anoptional third transcriptional unit, or on an additional transcriptionalunit that is operably linked to the first inducible promoter. Themammalian cell, which can be a packaging cell, can be a 293 cell.

In any of the methods provided herein that include a mammalian packagingcell, including a replication incompetent recombinant retroviralparticle packaging system aspect, or a method for making a replicationincompetent recombinant retroviral particle, for example, the mammaliancell, for example the packaging cell can include a nucleic acid sequenceencoding Vpx, for example on the second or an optional thirdtranscriptional unit, or on an additional transcriptional unit that isoperably linked to the first inducible promoter. The mammalian cell,which can be a packaging cell, can be a 293 cell.

In any of the methods provided herein that include a mammalian packagingcell, including a replication incompetent recombinant retroviralparticle packaging system aspect, or a method for making a replicationincompetent recombinant retroviral particle, a first ligand can berapamycin and a second ligand can be tetracycline or doxorubicin or thefirst ligand can be tetracycline or doxorubicin and the second ligandcan be rapamycin.

In some aspects, provided herein is a cell that has been transduced withany of the replication incompetent recombinant retroviral particlesprovided herein. The cell can be, for example, a lymphocyte, such as a Tcell or NK cell. The cell in illustrative embodiments, is a human cell.

In one aspect provided herein, is a method of expanding modified T cellsand/or NK cells in a subject, said method comprising:

-   -   A. contacting isolated resting T cells and/or resting NK cells        obtained from said subject with the replication incompetent        recombinant retroviral particle of any of the embodiments        disclosed herein;    -   B. introducing the genetically modified T cells and/or NK cells        into the subject; and    -   C. providing an effective amount of acyclovir, an acyclovir        prodrug, penciclovir, or a penciclovir prodrug to said subject,        wherein said modified T cells and/or NK cells proliferate in        said subject upon administration of acyclovir, an acyclovir        prodrug, penciclovir, or a penciclovir prodrug, thereby        expanding the modified T cells and/or NK cells in the subject.

In another aspect, provided herein is a method of stopping theexpansion, engraftment, and/or persistence of modified T cells and/or NKcells in a subject, said method comprising:

-   -   A. contacting isolated quiescent T cell and/or NK cells obtained        from said subject with the replication incompetent recombinant        retroviral particles of any of the embodiments disclosed herein;    -   B. introducing the modified T cell and/or NK cells into the        subject;    -   C. administering an effective amount of acyclovir, an acyclovir        prodrug, penciclovir, or a penciclovir prodrug to said subject        to expand the modified T cell and/or NK cells in the subject,        wherein said modified T cell and/or NK cells proliferate in said        subject upon administration of acyclovir, an acyclovir prodrug,        penciclovir, or a penciclovir prodrug, thereby expanding the        modified PBLs in the subject; and    -   D. stopping administration of acyclovir, an acyclovir prodrug,        penciclovir, or a penciclovir prodrug, wherein said modified T        cell and/or NK cells stop proliferating in said subject upon        stopping administration of acyclovir, an acyclovir prodrug,        penciclovir, or a penciclovir prodrug, thereby controlling the        expansion, expansion, and/or persistence of the modified T cell        and/or NK cells in the subject.

In another aspect, provided herein is a method of treating cancer in asubject, said method comprising:

-   -   A. contacting isolated quiescent T cells and/or NK cells        obtained from said subject with the replication incompetent        recombinant retroviral particles according to any of the        embodiments disclosed herein;    -   B. introducing the genetically modified T cells and/or NK cells        into the subject; and    -   C. administering an effective amount of acyclovir, an acyclovir        prodrug, penciclovir, or a penciclovir prodrug to said subject        to expand the modified T cell and/or NK cells in the subject,        wherein said modified T cell and/or NK cells proliferate in said        subject upon administration of acyclovir, an acyclovir prodrug,        penciclovir, or a penciclovir prodrug, and wherein the chimeric        antigen receptor in said modified T cell and/or NK cells binds        cancer cells in said subject, thereby treating cancer in the        subject.

In another aspect, provided herein is a transduced T cell and/or NKcell, comprising a recombinant polynucleotide comprising one or moretranscriptional units operatively linked to a promoter active in T cellsand/or NK cells, wherein the one or more transcriptional units encode afirst engineered signaling polypeptide regulated by a control element,wherein said first engineered signaling polypeptide comprises aconstitutively active IL-7 receptor mutant, and wherein the controlelement is capable of binding, and/or designed and/or configured tobind, to a compound in vivo.

In another aspect, provided herein is a retroviral packaging system,comprising:

-   -   a mammalian cell comprising:    -   A. a first transactivator expressed from a constitutive promoter        and capable of binding a first ligand and a first inducible        promoter for affecting expression of a nucleic acid sequence        operably linked thereto in the presence versus absence of the        first ligand;    -   B. a second transactivator capable of binding a second ligand        and a second inducible promoter, and affecting expression of a        nucleic acid sequence operably linked thereto in the presence        versus absence of the second ligand; and    -   C. a packageable RNA genome for a retroviral particle, wherein        the first transactivator regulates expression of the second        transactivator and a retroviral REV protein, wherein the second        transactivator regulates expression of a gag polypeptide, a pol        polypeptide, and one or more pseudotyping elements capable of        binding to a target cell and facilitating membrane fusion        thereto, and wherein the retroviral proteins are derived from a        retrovirus. Embodiments of this aspect, can include any of the        embodiments provided herein for the recited elements in other        aspects.

In another aspect, provided herein is a method for making a replicationincompetent recombinant retroviral particle, comprising:

-   -   A. culturing a population of packaging cells to accumulate a        first transactivator, wherein the packaging cells comprise the        first transactivator expressed from a first constitutive        promoter, wherein the first transactivator is capable of binding        a first ligand and a first inducible promoter for affecting        expression of a nucleic acid sequence operably linked thereto in        the presence versus absence of the first ligand, and wherein        expression of a second transactivator and a retroviral REV        protein is regulated by the first transactivator;    -   B. incubating the population of packaging cells comprising        accumulated first transactivator in the presence of the first        ligand to accumulate the second transactivator and the        retroviral REV protein, wherein the second transactivator is        capable of binding a second ligand and a second inducible        promoter for affecting expression of a nucleic acid sequence        operably linked thereto in the presence versus absence of the        second ligand; and    -   C. incubating the population of packaging cells comprising        accumulated second transactivator and retroviral REV protein in        the presence of the second ligand thereby inducing expression of        a gag polypeptide, a pol polypeptide, and one or more        pseudotyping elements, thereby making the replication        incompetent recombinant retroviral particle, wherein a        packageable RNA genome is encoded by a polynucleotide operably        linked to a third promoter, wherein said third promoter is        either constitutively active or inducible by either the first        transactivator or the second transactivator, and wherein the one        or more pseudotyping elements are capable of binding to a target        cell and/or facilitating membrane fusion of the replication        incompetent recombinant retroviral particle thereto.

In some embodiments of the retroviral packaging system and method formaking a replication incompetent recombinant retroviral particlesprovided herein, the mammalian cell further comprises an activationelement capable of binding to and activating a target cell, typically alymphocyte, such as an NK cell, or in illustrative embodiments a T cell,and the first transactivator regulates the expression of the activationelement. The activation element is on the surface of the replicationincompetent recombinant retroviral particle and wherein the activationelement can include: a membrane-bound polypeptide capable of binding toCD3; and/or a membrane-bound polypeptide capable of binding to CD28. Themembrane-bound polypeptide capable of binding to CD3 is a polypeptidecapable of binding to CD3 that is fused to a heterologous GPI anchorattachment sequence and the membrane-bound polypeptide capable ofbinding to CD28 is a polypeptide capable of binding to CD28 that isfused to a heterologous GPI anchor attachment sequence. Themembrane-bound polypeptide capable of binding to CD28 in someembodiments comprises CD80, CD86, or a functional fragment thereof thatis capable of inducing CD28-mediated activation of Akt, such as theextracellular domain of CD80. In other embodiments, membrane-boundpolypeptide capable of binding CD3 is an anti-CD3 scFv or an anti-CD3scFvFc bound to a CD14 GPI anchor attachment sequence, and wherein themembrane-bound polypeptide capable of binding to CD28 is CD80, or anextracellular fragment thereof, bound to a CD16B GPI anchor attachmentsequence.

In some embodiments of the retroviral packaging system and method formaking a replication incompetent recombinant retroviral particle aspectsprovided herein, the mammalian cell further comprises a membrane-boundcytokine, and the first transactivator regulates the expression of themembrane-bound cytokine. The membrane-bound cytokine can be, forexample, IL-7, IL-15, or an active fragment thereof. The membrane-boundcytokine in embodiments can be a fusion polypeptide of IL-7, or anactive fragment thereof, and DAF. For example, the fusion polypeptidecan comprise the DAF signal sequence and IL-7 without its signalsequence, followed by residues 36-525 of DAF.

In some embodiments of the retroviral packaging system and method formaking a replication incompetent recombinant retroviral particle aspectsprovided herein, the mammalian cell comprises associated with itsmembrane, an activation element comprising an anti-CD3 scFV or ananti-CD3 scFvFc bound to a CD14 GPI anchor attachment sequence and aCD80 bound, or an extracellular fragment thereof to a CD16B GPI anchorattachment sequence; and membrane-bound cytokine comprising a fusionpolypeptide of IL-7, or an active fragment thereof, and DAF comprising aGPI anchor attachment sequence, and wherein the first transactivatorregulates the expression of each of the activation element andmembrane-bound cytokine. In some embodiments, the IL-7, or an activefragment thereof, and DAF fusion, the anti-CD3 scFV or an anti-CD3scFvFc, and the CD80, or extracellular fragment thereof, each comprisesa DAF signal sequence.

In some embodiments of the retroviral packaging system and method formaking a replication incompetent recombinant retroviral particle aspectsprovided herein, the mammalian cell further comprises a Vpu polypeptide.In some embodiments of the retroviral packaging system and method formaking a replication incompetent recombinant retroviral particle aspectsprovided herein, the mammalian cell further comprises a Vpu polypeptideand a Vpx polypeptide. In some embodiments of the retroviral packagingsystem and method for making a replication incompetent recombinantretroviral particle aspects provided herein, the mammalian cell furthercomprises a Vpx polypeptide. In these or other embodiments, the one ormore pseudotyping elements comprise one or more viral polypeptidesrecognized by T cells. The one or more pseudotyping elements cancomprise a Measles Virus F polypeptide, a Measles Virus H polypeptide,and/or a fragment thereof. In certain illustrative embodiments, the oneor more pseudotyping elements are cytoplasmic domain deletion variantsof a measles virus F polypeptide and/or a measles virus H polypeptide.

In some embodiments of the retroviral packaging system and method formaking a replication incompetent recombinant retroviral particle aspectsprovided herein, the packageable RNA genome is encoded by apolynucleotide operably linked to a third promoter, wherein said thirdpromoter is either constitutively active or inducible by either thefirst transactivator or the second transactivator. In illustrativeembodiments, the packageable RNA genome is encoded by a polynucleotideoperably linked to a third promoter, wherein said third promoter isinducible by the second transactivator.

In some embodiments of the retroviral packaging system and method formaking a replication incompetent recombinant retroviral particle aspectsprovided herein, the packageable RNA genome further comprises, from 5′to 3′:

-   -   a) a 5′ long terminal repeat, or active fragment thereof;    -   b) a nucleic acid sequence encoding a retroviral cis-acting RNA        packaging element;    -   c) a nucleic acid sequence encoding a first target polypeptide        and an optional second target polypeptide;    -   d) a fourth promoter operably linked to the first target        polypeptide and the optional second polypeptide, wherein said        fourth promoter is active in the target cell but not active in        the packaging cell line; and    -   e) a 3′ long terminal repeat, or active fragment thereof.

In some embodiments of the retroviral packaging system and method formaking a replication incompetent recombinant retroviral particle aspectsprovided herein including the construct immediately above, the thirdpromoter promotes transcription or expression in the opposite directionfrom transcription or expression promoted from the fourth promoter.

In some embodiments of the retroviral packaging system and method formaking a replication incompetent recombinant retroviral particle aspectsprovided herein, the packageable RNA genome encodes the replicationincompetent recombinant retroviral particle of any embodiment disclosedin this disclosure, wherein the first target polypeptide and the secondtarget polypeptide are the first engineered signaling polypeptide andthe second engineered signaling polypeptide, respectively. In someembodiments, for example, the packageable RNA genome further comprises acontrol element operably linked to the nucleic acid encoding the firstengineered signaling polypeptide or the second engineered signalingpolypeptide. The control element in illustrative embodiments is ariboswitch. The riboswitch in illustrative embodiments is capable ofbinding a compound and the compound that binds the control element is anucleoside analog, and the nucleoside analog can be an antiviral drug,for example acyclovir or penciclovir.

In some embodiments of the retroviral packaging system and method formaking a replication incompetent recombinant retroviral particle aspectsprovided herein, the packageable RNA genome further comprises an introncomprising a polynucleotide encoding an inhibitory RNA molecules, suchas, e.g., a miRNA or shRNA. The intron can be adjacent to and downstreamof the fourth promoter.

In some embodiments of the retroviral packaging system and method formaking a replication incompetent recombinant retroviral particle aspectsprovided herein, the target cell can be a T cell and/or an NK cell.

In some embodiments of the retroviral packaging system and method formaking a replication incompetent recombinant retroviral particle aspectsprovided herein, the one or more pseudotyping elements comprise avesicular stomatitis virus envelope protein (VSV-G), a feline endogenousvirus (RD114) envelope protein, an oncoretroviral amphotropic envelopeprotein, or an oncoretroviral ecotropic envelope protein, or functionalfragments thereof.

In some embodiments of the retroviral packaging system and method formaking a replication incompetent recombinant retroviral particle aspectsprovided herein, the packageable RNA genome is 11,000 KB or less or10,000 KB or less in size. In some embodiments of the retroviralpackaging system and method for making a replication incompetentrecombinant retroviral particle aspects provided herein, the firsttarget polypeptide comprises a first engineered signaling polypeptideand wherein said first engineered signaling polypeptide comprises atleast one lymphoproliferative element, and the second target polypeptidecomprises a second engineered signaling polypeptide including a CAR.

In one aspect, provided herein is an isolated polynucleotide forregulating expression of a target polynucleotide, comprising:

-   -   a polynucleotide encoding a target polynucleotide operably        linked to a promoter and a riboswitch, wherein the riboswitch        comprises:        -   a.) an aptamer domain capable of binding a nucleoside            analogue antiviral drug and having reduced binding to            guanine or 2′-deoxyguanosine relative to the nucleoside            analogue antiviral drug; and        -   b.) a function switching domain capable of regulating            expression of the target polynucleotide, wherein binding of            the nucleoside analogue by the aptamer domain induces or            suppresses the expression regulating activity of the            function switching domain, thereby regulating expression of            the target polynucleotide.

In illustrative embodiments of any of the methods and compositionsprovided herein that include the control element can be a polynucleotidecomprising a riboswitch. The riboswitch can be capable of binding anucleoside analog and the compound that binds the control element is thenucleoside analog. The nucleoside analog can be an antiviral agent. Theantiviral agent can be acyclovir or penciclovir. The riboswitch canpreferentially bind acyclovir over penciclovir or preferentially bindpenciclovir over acyclovir. The riboswitch can have reduced binding tothe nucleoside analogue antiviral drug at temperatures above 37° C.,37.5° C., 38° C., 38.5° C., or 39° C., for example, above 39° C. Theriboswitch can be between 35, 40, 45, and 50 nucleotides in length onthe low end of the range and 60, 65, 70, 75, 80, 85, 90, 95, and 100nucleotides in length on the high end of the range, for example, between45 and 80 nucleotides in length. In illustrative embodiments of any ofthe methods and compositions provided herein that include theriboswitch, the target polynucleotide that is regulated by theriboswitch can include a region encoding a miRNA, an shRNA, and/or apolypeptide. The target polynucleotide can encode a lymphoproliferativeelement. The target polynucleotide can be operably linked to a promoter.The target polynucleotide can include a region encoding a polypeptideand the polypeptide can include a chimeric antigen receptor comprisingan antigen-specific targeting region, a transmembrane domain, and anintracellular activating domain. In illustrative embodiments of any ofthe methods and compositions provided herein that include theriboswitch, the function switching domain can regulate an internalribosome entry site, pre-mRNA splice donor accessibility in the viralgene construct, translation, termination of transcription, transcriptdegradation, miRNA expression, or shRNA expression, thereby regulatingexpression of the target polynucleotide. The riboswitch can include aribozyme. In illustrative embodiments of any of the methods andcompositions provided herein that include the riboswitch, the isolatedpolynucleotide can be a molecular cloning vector or an expressionvector. In illustrative embodiments of any of the methods andcompositions provided herein that include the riboswitch, the isolatedpolynucleotide can be integrated into a retroviral genome or into amammalian chromosome, or fragment thereof. In certain embodiments ofthis and any of the aspects and embodiments herein that comprise ariboswitch, the riboswitch regulates pre-mRNA splicing. For example, theriboswitch can comprise a branchpoint sequence, typically between twoexons on a polynucleotide and/or transcriptional unit herein. As such,binding of a nucleoside analogue antiviral compound or drug to theriboswitch regulates exon splicing. Such embodiments can include apolynucleotide comprising a first exon, a second exon, and a riboswitchbetween the first exon and the second exon, wherein the riboswitchcomprises a branchpoint sequence, and wherein binding of a nucleosideanalogue antiviral compound or drug (e.g. acyclovir) to the riboswitchregulates exon splicing of the first and second exon. In otherembodiments of this and any of the aspects and embodiments herein thatcomprise a riboswitch, the riboswitch can control gene expression byregulating pre-mRNA transplicing. In such embodiments, the riboswitch islocated within a transplicing intron. For example, one aspect caninclude a polynucleotide that comprises a first exon, a second exon, anda riboswitch between the first exon and the second exon, wherein theriboswitch comprises a branchpoint sequence, and wherein binding ofacyclovir to the riboswitch regulates exon splicing of the first andsecond exon.

Another aspect provided herein, is a method for genetically modifyingand expanding lymphocytes of a subject, comprising:

-   -   A. collecting blood from the subject;    -   B. contacting T cells and/or NK cells from the blood of the        subject ex vivo with replication incompetent recombinant        retroviral particles comprising:        -   i. a pseudotyping element on its surface that is capable of            binding to a T cell and/or NK cell and facilitating membrane            fusion of the replication incompetent recombinant retroviral            particle thereto, wherein said pseudotyping element            comprises cytoplasmic domain deletion variants of a measles            virus F polypeptide and/or a measles virus H polypeptide;        -   ii. a polypeptide capable of binding to CD3 and a            polypeptide capable of binding to CD28, wherein said            polypeptides are expressed on the surface of a replication            incompetent recombinant retroviral particle and are capable            of binding to a T cell and/or a NK cell and further wherein            said polypeptides are not encoded by a polynucleotide in the            replication incompetent recombinant retroviral particle; and        -   iii. a polynucleotide comprising one or more transcriptional            units operatively linked to a promoter active in T cells            and/or NK cells, wherein the one or more transcriptional            units encode a first engineered signaling polypeptide            comprising a constitutively active IL-7 receptor mutant and            a second engineered signaling polypeptide comprising a            chimeric antigen receptor comprising an antigen-specific            targeting region (ASTR), a transmembrane domain, and an            intracellular activating domain, wherein expression of the            IL-7 receptor mutant is regulated by a riboswitch that binds            a nucleoside analog antiviral drug, wherein binding of the            nucleoside analog antiviral drug to the riboswitch increases            expression of the IL-7 receptor mutant, and wherein said            contacting results in at least some of the resting T cells            and/or NK cells becoming genetically modified;    -   C. reintroducing the genetically modified T cells and/or NK        cells into the subject; and    -   D. exposing the genetically modified T cells and/or NK cells in        vivo to the nucleoside analog antiviral drug to promote        expansion of the T cells and/or NK cells, wherein the method        between the collecting blood and the reintroducing the        genetically modified T cells and/or NK cells is performed in no        more than 24 hours and/or without requiring prior ex vivo        stimulation, thereby genetically modifying and expanding        lymphocytes of the subject.

In illustrative embodiments of this method aspect, the retroviralparticle is a lentiviral particle. In another illustrative embodiment,the replication incompetent recombinant retroviral particle geneticallymodifies a T cell. In another illustrative embodiment, the polypeptidecapable of binding to CD3 and the polypeptide capable of binding to CD28are each fused to a heterologous GPI anchor attachment sequence. In someinstances, the polypeptide capable of binding to CD3 can be anti-CD3scFvFc or anti-CD3 scFv, and the polypeptide capable of binding to CD28can be CD80. The anti-CD3 scFvFc or anti-CD3 scFv and CD80 can each befurther fused to a DAF signal sequence. In another illustrativeembodiment, the replication incompetent recombinant retroviral particlesfurther comprise on their surface a fusion polypeptide comprising acytokine covalently attached to DAF. In some instances, the cytokine canbe IL-7 or IL-15, and the fusion polypeptide can comprise the DAF signalsequence, IL-7 without its signal sequence, and a fragment of DAFcomprising a GPI anchor attachment sequence.

In another illustrative embodiment of this method aspect immediatelyabove, the riboswitch further controls expression of the chimericantigen receptor in a manner regulated by binding of the riboswitch tothe nucleoside analog antiviral drug, which in some instances isacyclovir and/or penciclovir. In another embodiment, the constitutivelyactive IL-7 can be replaced with a miRNA or shRNA or nucleic acidsencoding an miRNA or shRNA and IL-7 can be present. In some instances,the miRNA or shRNA can be encoded by nucleic acids within an intron.

Another aspect provided herein is a replication incompetent recombinantretroviral particle, comprising:

-   -   A. a pseudotyping element on its surface that is capable of        binding to a T cell and/or NK cell and facilitating membrane        fusion of the replication incompetent recombinant retroviral        particle thereto, wherein said pseudotyping element comprises        cytoplasmic domain deletion variants of a measles virus F        polypeptide and/or a measles virus H polypeptide;    -   B. a polynucleotide comprising one or more transcriptional units        operatively linked to a promoter active in T cells and/or NK        cells, wherein the one or more transcriptional units encode a        first engineered signaling polypeptide comprising a chimeric        antigen receptor comprising an antigen-specific targeting        region, a transmembrane domain, and an intracellular activating        domain, and a second engineered signaling polypeptide comprising        a constitutively active IL-7 receptor mutant; wherein expression        of the IL-7 receptor mutant is regulated by a riboswitch that        binds a nucleoside analog antiviral drug, wherein binding of the        nucleoside analog antiviral drug to the riboswitch increases        expression of the IL-7 receptor mutant; and    -   C. a polypeptide capable of binding to CD3 and a polypeptide        capable of binding to CD28, wherein said polypeptides are        expressed on the surface of a replication incompetent        recombinant retroviral particle; are capable of binding to a T        cell and/or NK cell; and are not encoded by a polynucleotide in        the replication incompetent recombinant retroviral particle.

In illustrative embodiments of any replication incompetent recombinantretroviral particle aspect, the retroviral particle is a lentiviralparticle. In other illustrative embodiments of the method, thepolypeptide capable of binding to CD3 and the polypeptide capable ofbinding to CD28 are each fused to a heterologous GPI anchor attachmentsequence. In some instances, the polypeptide capable of binding to CD3can be anti-CD3 scFvFc or anti-CD3 scFv, and the polypeptide capable ofbinding to CD28 can be CD80. The anti-CD3 scFvFc or anti-CD3 scFv andCD80 can each be further fused to a DAF signal sequence. In anotherillustrative embodiment, the replication incompetent recombinantretroviral particles further comprise on their surface a fusionpolypeptide comprising a cytokine covalently attached to DAF. In someinstances, the cytokine can be IL-7 or IL-15, and the fusion polypeptidecan comprise the DAF signal sequence, IL-7 without its signal sequence,and a fragment of DAF comprising a GPI anchor attachment sequence.

In another illustrative embodiment of any replication incompetentrecombinant retroviral particle aspect herein, the riboswitch furthercontrols expression of the chimeric antigen receptor in a mannerregulated by binding of the riboswitch to the nucleoside analogantiviral drug, which in some instances is acyclovir and/or penciclovir.In another embodiment, the constitutively active IL-7 can be replacedwith a miRNA or shRNA or nucleic acids encoding an miRNA or shRNA andIL-7 can be present. The miRNA or shRNA can be encoded by nucleic acidswithin an intron.

Another aspect provided herein is a method for making a replicationincompetent recombinant retroviral particle, comprising:

-   -   A. culturing a population of packaging cells to accumulate a        first transactivator, wherein the packaging cells comprise the        first transactivator expressed from a constitutive promoter,        wherein the first transactivator is capable of binding a first        ligand and a first inducible promoter for affecting expression        of a nucleic acid sequence operably linked thereto in the        presence versus absence of the first ligand, and wherein        expression of a second transactivator and a retroviral REV        protein is regulated by the first transactivator;    -   B. incubating the population of packaging cells comprising        accumulated first transactivator in the presence of the first        ligand to accumulate the second transactivator and the        retroviral REV protein and an activation element typically on        their surface, comprising a polypeptide capable of binding to        CD3 and a polypeptide capable of binding to CD28, wherein the        second transactivator is capable of binding a second ligand and        a second inducible promoter for affecting expression of a        nucleic acid sequence operably linked thereto in the presence        versus absence of the second ligand; and    -   C. incubating the population of packaging cells comprising        accumulated second transactivator and retroviral REV protein in        the presence of the second ligand thereby inducing expression of        a gag polypeptide, a pol polypeptide and a pseudotyping element        capable of binding to a T cell and/or an NK cell and        facilitating membrane fusion of the replication incompetent        recombinant retroviral particle thereto, wherein said        pseudotyping element comprises cytoplasmic domain deletion        variants of a measles virus F polypeptide and/or a measles virus        H polypeptide, wherein a packageable RNA genome is encoded by a        polynucleotide operably linked to a third promoter and wherein        said promoter is inducible by the second transactivator, wherein        the packageable RNA genome comprises, from 5′ to 3′:        -   i. a 5′ long terminal repeat, or active fragment thereof;        -   ii. a nucleic acid sequence encoding a retroviral cis-acting            RNA packaging element;        -   iii. a nucleic acid sequence encoding a first engineered            signaling polypeptide comprising a chimeric antigen receptor            and a second engineered signaling polypeptide comprising a            constitutively active IL-7 receptor mutant separated by a            cleavage signal;        -   iv. a fourth promoter that is active in the T cell and/or            the NK cell; and        -   v. a 3′ long terminal repeat, or active fragment thereof,            and wherein the packageable RNA genome further comprises a            riboswitch that binds a nucleoside analog antiviral drug,            wherein binding of the riboswitch to the nucleoside analog            antiviral drug to the riboswitch increases expression of the            IL-7 receptor mutant, thereby making the replication            incompetent recombinant retroviral particle.

In an illustrative embodiment of the method, the riboswitch furthercontrols expression of the chimeric antigen receptor in a mannerregulated by binding of the riboswitch to the nucleoside analogantiviral drug. In another illustrative embodiment, the nucleosideanalog antiviral drug is acyclovir and/or penciclovir. In anotherillustrative embodiment, the packageable RNA genome further comprises arecognition domain, wherein the recognition domain comprises apolypeptide that is recognized by an antibody that recognizes EGFR or anepitope thereof. In another illustrative embodiment, the first ligand israpamycin and the second ligand is tetracycline or doxorubicin or thefirst ligand is tetracycline or doxorubicin and the second ligand israpamycin. In another illustrative embodiment, the packaging cellfurther comprises a nucleic acid sequence encoding Vpu on the second oran optional third transcriptional unit, or on an additionaltranscriptional unit that is operably linked to the first induciblepromoter. In another illustrative embodiment, the packaging cell furthercomprises a nucleic acid sequence encoding a Vpu polypeptide andoptionally a Vpx polypeptide on the second or an optional thirdtranscriptional unit, or on an additional transcriptional unit that isoperably linked to the first inducible promoter. In another illustrativeembodiment, the packaging cell further comprises a nucleic acid sequenceencoding Vpx on the second or an optional third transcriptional unit, oron an additional transcriptional unit that is operably linked to thefirst inducible promoter. In another illustrative embodiment, thepolypeptide capable of binding to CD3 and the polypeptide capable ofbinding to CD28 are each fused to a heterologous GPI anchor attachmentsequence. In some instances, the polypeptide capable of binding to CD3can be anti-CD3 scFvFc or anti-CD3 scFv, or anti-CD3 scFv, and thepolypeptide capable of binding to CD28 can be CD80. The anti-CD3 scFvFcor anti-CD3 scFv and CD80 can each be further fused to a DAF signalsequence. In another illustrative embodiment, expression of a fusionpolypeptide comprising a cytokine covalently attached to DAF is alsoinduced. In some instances, the cytokine can be IL-7 or IL-15, and thefusion polypeptide can comprise the DAF signal sequence, IL-7 withoutits signal sequence, and a fragment of DAF comprising a GPI anchorattachment sequence. In another illustrative embodiment, the riboswitchfurther controls expression of the chimeric antigen receptor in a mannerregulated by binding of the riboswitch to the nucleoside analogantiviral drug, which in some instances is acyclovir and/or penciclovir.In another embodiment, the constitutively active IL-7 can be replacedwith a miRNA or shRNA or nucleic acids encoding an miRNA or shRNA andIL-7 can be present. The miRNA or shRNA can be encoded by nucleic acidswithin an intron. In an illustrative embodiment, the retroviral particleis a lentiviral particle.

Provided in another aspect herein is a genetically modified lymphocytecomprising:

-   -   A. a first engineered signaling polypeptide comprising a        constitutively active IL-7 receptor mutant; and    -   B. a second engineered signaling polypeptide comprising a        chimeric antigen receptor comprising an antigen-specific        targeting region (ASTR), a transmembrane domain, and an        intracellular activating domain.

In illustrative embodiments of the genetically modified lymphocyteaspect above, the genetically modified lymphocyte is a T cell and/or anNK cell. In certain embodiments, the lymphocyte is a T cell. In anotherillustrative embodiment, expression of said first engineered signalingpolypeptide and/or said second engineered signaling polypeptide isregulated by a riboswitch that binds a nucleoside analog antiviral drug,wherein binding of the nucleoside analog antiviral drug to theriboswitch increases expression of the IL-7 receptor mutant. In anotherembodiment, the genetically modified lymphocytes express at least one(e.g. two) inhibitory RNA molecules, such as, e.g. a miRNA or an shRNA.The inhibitory RNA molecules can further be encoded by nucleic acidswithin an intron.

Provided in another aspect herein is a genetically modified T celland/or NK cell comprising:

-   -   a. a first engineered signaling polypeptide comprising at least        one lymphoproliferative element; and    -   b. a second engineered signaling polypeptide comprising a        chimeric antigen receptor comprising an antigen-specific        targeting region (ASTR), a transmembrane domain, and an        intracellular activating domain.

In illustrative embodiments of the genetically modified T cell and/or NKcell aspect, the lymphoproliferative element is constitutively active,and in some instances, is a constitutively active mutated IL-7 receptoror a fragment thereof. In another illustrative embodiment, expression ofthe first engineered signaling polypeptide and/or the second engineeredsignaling polypeptide is regulated by a control element. In someinstances, the control element is a polynucleotide comprising ariboswitch. In some instances, the riboswitch is capable of binding anucleoside analog and when the nucleoside analog is present, the firstengineered signaling polypeptide and/or the second engineeredpolypeptide are expressed. In other illustrative embodiments, thegenetically modified T cell and/or NK cell has on its surface anactivation element, a pseudotyping element, and/or a membrane-boundcytokine. In some instances, the activation element comprises amembrane-bound polypeptide capable of binding to CD3; and/or amembrane-bound polypeptide capable of binding to CD28. In a certainembodiment, the activation element comprises anti-CD3 scFV or ananti-CD3 scFvFc fused to a heterologous GPI anchor attachment sequenceand/or CD80 fused to a heterologous GPI anchor attachment sequence. Inan illustrative embodiment, the pseudotyping element comprises a MeaslesVirus F polypeptide, a Measles Virus H polypeptide, and/or cytoplasmicdomain deletion variants of a measles virus F polypeptide and/or ameasles virus H polypeptide. In other embodiments, the membrane-boundcytokine is a fusion polypeptide comprising IL-7, or a fragment thereof,fused to DAF, or a fragment thereof comprising a GPI anchor attachmentsequence.

In one aspect, provided herein is a method for genetically modifying andexpanding lymphocytes of a subject, comprising:

-   -   A. contacting resting T cells and/or NK cells of the subject ex        vivo, typically without requiring prior ex vivo stimulation,        with replication incompetent recombinant retroviral particles        comprising:        -   i. a pseudotyping element on its surface that is capable of            binding to a T cell and/or NK cell and facilitating membrane            fusion of the replication incompetent recombinant retroviral            particle thereto; and        -   ii. a polynucleotide comprising one or more transcriptional            units operatively linked to a promoter active in T cells            and/or NK cells, wherein the one or more transcriptional            units encode a first engineered signaling polypeptide            regulated by a control element, wherein said first            engineered signaling polypeptide comprises at least one            lymphoproliferative element and optionally encode a second            engineered signaling polypeptide optionally regulated by a            control element, wherein the second engineered signaling            polypeptide comprises an intracellular activating domain and            optionally other components of a CAR, wherein said            contacting facilitates transduction of at least some of the            resting T cells and/or NK cells by the replication            incompetent recombinant retroviral particles, thereby            producing genetically modified T cells and/or NK cells;    -   B. introducing the genetically modified T cells and/or NK cells        into the subject; and exposing the genetically modified T cells        and/or NK cells in vivo to a compound that acts as the control        element to affect expression of the first engineered signaling        polypeptide and promote expansion, engraftment, and/or        persistence of the lymphocytes in vivo, thereby genetically        modifying and expanding lymphocytes of the subject.

In illustrative embodiments, the transduction is carried out without exvivo stimulation. In illustrative embodiments, the compound is amolecular chaperone, such as a small molecular chaperone. Inillustrative embodiments, binding of the molecular chaperone to thelymphoproliferative element increases the proliferative activity of thelymphoproliferative element. The molecular chaperone can be administeredto the subject before the blood is collected, during the contacting,and/or after the T cells and/or NK cells are introduced into thesubject. It will be understood with this aspect where the compound isthe control element, that such compound typically is capable of bindingto a lymphoproliferative element and/or a component of a CAR, and doesbind to such lymphoproliferative element or CAR component duringperformance of the method. Other embodiments and teaches related tomethods provided herein that include transfecting a T cell and/or an NKcell with a replication incompetent recombinant retroviral particle,apply to this aspect, including a molecular chaperone embodiment, aswell.

In another aspect, provided herein is a method for selecting amicroenvironment restricted antigen-specific targeting region,comprising panning a polypeptide display library by: a. subjectingpolypeptides of the polypeptide display library to a binding assay undera normal physiological condition and a binding assay under an aberrantcondition; and b. selecting a polypeptide which exhibits an increase inbinding activity at the aberrant condition compared to the physiologicalcondition, thereby selecting the microenvironment restricted antigenspecific targeting region.

In another aspect, provided herein is a method for isolating amicroenvironment restricted antigen-specific targeting region,comprising:

-   -   panning a polypeptide library by:        -   a) contacting the polypeptide library under aberrant            conditions with a target antigen bound to a solid support,            wherein clones expressing polypeptides that bind the target            antigen remain bound to the solid support through the target            antigen;        -   b) incubating the solid supports with bound polypeptides            under physiological conditions; and        -   c) collecting clones that elute from the solid support under            the physiological conditions, thereby isolating the            microenvironment restricted antigen-specific targeting            region.

In another aspect, provided herein is a chimeric antigen receptor forbinding a target antigen, comprising:

-   -   a) at least one microenvironment restricted antigen specific        targeting region selected by panning a polypeptide library and        having an increase in activity in a binding assay at an aberrant        condition compared to a normal physiological condition;    -   b) a transmembrane domain; and    -   c). an intracellular activating domain.

In another aspect, provided herein is a chimeric antigen receptor forbinding a target antigen, comprising:

-   -   a) a microenvironment restricted antigen-specific targeting        region that exhibits an increase in binding to the target        antigen in an aberrant condition compared to a normal        physiological environment, wherein the antigen-specific        targeting region binds to the target;    -   b) a transmembrane domain; and    -   c) an intracellular activating domain.

In illustrative embodiments of any of the methods and compositionsprovided herein that include a microenvironment restricted antigenspecific targeting region (ASTR), the ASTR can have at least a 5%, 10%,15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% increase inbinding affinity to the target antigen in the assay at the aberrantcondition compared to the normal condition. The aberrant conditions canbe hypoxia, an acidic pH, a higher concentration of lactic acid, ahigher concentration of hyaluronan, a higher concentration of albumin, ahigher concentration of adenosine, a higher concentration ofR-2-hydroxyglutarate, a higher concentration of PAD enzymes, a higherpressure, a higher oxidation, and a lower nutrient availability. Themicroenvironment restricted ASTR can exhibit an increase in antigenbinding at a pH of 6.7 as compared to a pH of 7.4. The microenvironmentrestricted ASTR can exhibit an increase in antigen binding in a tumorenvironment and/or in an in vitro tumor surrogate assay condition,relative to a corresponding physiological condition. The target can be4-1BB, ST4, adenocarcinoma antigen, alpha-fetoprotein, AXL, BAFF,B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX),C-MET, CCR4, CD 152, CD 19, CD20, CD200, CD22, CD221, CD23 (IgEreceptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52,CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DRS, EGFR, EpCAM, CD3, FAP,fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside,glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptorkinase, IGF-1 receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6, insulin-likegrowth factor I receptor, integrin nSP1, integrin nvP3, MORAb-009,MS4A1, MUC1, mucin CanAg, Nglycolylneuraminic acid, NPC-1C, PDGF-R a,PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, ROR1,ROR2 SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-P,TRAIL-R1, TRAIL-R2, tumor antigen CTAA16. 88, VEGF-A, VEGFR-1, VEGFR2,and vimentin. The ASTR can be an antibody, an antigen, a ligand, areceptor binding domain of a ligand, a receptor, a ligand binding domainof a receptor, or an affibody. The ASTR can be a full-length antibody, asingle-chain antibody, an Fab fragment, an Fab′ fragment, an (Fab′)₂fragment, an Fv fragment, and a divalent single-chain antibody or adiabody. The ASTR can include a heavy chain and a light chain from anantibody. The antibody can be a single-chain variable fragment. In someembodiments, the heavy and light chains can be separated by a linker,wherein the linker is between 6 and 100 amino acids in length. In someembodiments, the heavy chain can be positioned N-terminal to the lightchain on the chimeric antigen receptor and in some embodiments the lightchain can be positioned N-terminal to the heavy chain on the chimericantigen receptor.

In illustrative embodiments of any of the methods that include apolypeptide display library, the polypeptide display library can be aphage display library or a yeast display library. The polypeptidedisplay library can be an antibody display library. The antibody displaylibrary can be a human or humanized antibody display library. Theantibody display library can be a naïve library. The methods can includeinfecting bacterial cells with the collected phage to generate a refinedphage display library, and repeating the contacting, incubating, andcollecting for 1 to 1000 cycles, using the refined phage display librarygenerated from a previous cycle.

In illustrative embodiments of any of the methods provided herein thatinclude isolating or selecting a microenvironment restricted ASTR, themethod can include determining the nucleotide sequence of apolynucleotide encoding the microenvironment restricted antigen-specifictargeting region, thereby determining the polypeptide sequence of themicroenvironment restricted ASTR. The methods can include making amicroenvironment restricted biologic chimeric antigen receptor bygenerating a polynucleotide that encodes a polypeptide comprising themicroenvironment restricted ASTR, a transmembrane domain, and anintracellular activating domain. The library can be a single chainantibody library.

The methods for isolating a microenvironment restricted ASTR can includethe panning is repeated for between 1 and 1000 times. The methods forisolating a microenvironment restricted ASTR can be performed withoutmutating polynucleotides encoding the isolated microenvironmentrestricted antigen-specific targeting region between rounds of panning.The methods for isolating a microenvironment restricted ASTR can beperformed by culturing, high fidelity amplifying, and/or dilutingpolynucleotides encoding antigen-specific targeting regions, or hostorganisms including the same, between rounds of panning. The methods caninclude, prior to repeating, mutagenizing the selected and/or isolatedmicroenvironment restricted antigen-specific targeting region. Themethods can include determining the sequence of the selected and/orisolated microenvironment restricted antigen-specific targeting region,and/or a polynucleotide encoding the same after one or more round ofpanning via long read DNA sequencing. The methods can includedetermining the sequence before and after expansion of the isolatedmicroenvironment restricted ASTR. The methods for isolating amicroenvironment restricted ASTR can be performed without repeating thepanning. The methods for isolating a microenvironment restricted ASTRcan be performed without mutating a polynucleotide encoding the isolatedmicroenvironment restricted ASTR after the microenvironment restrictedASTR is isolated.

In illustrative embodiments of any of the compositions provided hereinthat include a chimeric antigen receptor with a microenvironmentrestricted ASTR, the microenvironment restricted ASTR can be identifiedby panning an antibody library. In some embodiments, themicroenvironment restricted ASTR is identified by panning a phagedisplay or a yeast display library. In some embodiments, the chimericantigen receptor comprises a bispecific ASTR.

Provided herein in another aspect is a transduced T cell and/or NK cell,comprising a recombinant polynucleotide comprising one or moretranscriptional units operatively linked to a promoter active in T cellsand/or NK cells, wherein the one or more transcriptional units encode afirst engineered signaling polypeptide regulated by a control element,wherein said first engineered signaling polypeptide comprises aconstitutively active IL-7 receptor mutant, and wherein the controlelement is capable of binding to a compound in vitro or in vivo or isconfigured to bind a compound in vivo.

Provided herein in another aspect is a replication incompetentrecombinant retroviral particle, comprising a recombinant polynucleotidecomprising one or more transcriptional units operatively linked to apromoter active in T cells and/or NK cells, wherein the one or moretranscriptional units encode a first engineered signaling polypeptideregulated by a control element, which can be an in vivo control element,wherein said first engineered signaling polypeptide comprises aconstitutively active IL-7 receptor mutant, and wherein the controlelement is capable of binding to a compound in vivo or is configured tobind a compound in vivo.

Provided herein in another aspect is a method of transducing a T celland/or NK cell, comprising contacting a T cell and/or NK cell, with areplication incompetent recombinant retroviral particle comprising arecombinant polynucleotide comprising one or more transcriptional unitsoperatively linked to a promoter active in T cells and/or NK cells,wherein the one or more transcriptional units encode a first engineeredsignaling polypeptide regulated by a control element, wherein said firstengineered signaling polypeptide comprises a constitutively active IL-7receptor mutant, and wherein the in vivo control element is capable ofbinding to a compound in vivo or in vitro, under transductionconditions, thereby transducing the T cell and/or NK cell.

In illustrative embodiments of the transduced T cell and/or NK cellaspects, the replication incompetent recombinant retroviral particleaspects, and the method aspects, provided in the preceding paragraphs,the recombinant polynucleotide further comprises a transcriptional unitthat encodes a second engineered signaling polypeptide comprising afirst chimeric antigen receptor comprising an antigen-specific targetingregion (ASTR), a transmembrane domain, and an intracellular activatingdomain. In other illustrative embodiments, the lymphoproliferativeelement comprises a mutated IL-7 receptor or a fragment thereof. Inother illustrative embodiments, the control element is a polynucleotidecomprising a riboswitch. In some instances, the riboswitch is capable ofbinding a nucleoside analog and the compound that binds the controlelement is the nucleoside analog. In some instances, the nucleosideanalog is an antiviral agent such as for example acyclovir orpenciclovir. In certain embodiments, the antiviral agent is acyclovir.In other illustrative embodiments, the constitutively active IL-7receptor mutant is fused to EGFR or an epitope thereof. In otherillustrative embodiments, the constitutively active IL-7 receptor mutantcomprises an eTag. In other illustrative embodiments, the constitutivelyactive IL-7 receptor mutant comprises a PPCL insertion. In otherillustrative embodiments, the constitutively active IL-7 receptor mutantcomprises a PPCL insertion at a position equivalent to position 243 in awild-type human IL-8 receptor. In other illustrative embodiments, thetransduced T cell or NK cell is a transduced T cell.

In another aspect, provided herein is a method for modulating binding ofa microenvironment restricted biologic chimeric antigen receptor(MRB-CAR)-expressing T cell or NK cell to a cell expressing a cognateantigen of the MRB-CAR in a subject, including:

-   -   a. introducing a T cell and/or NK cell including a nucleic acid        encoding the MRB-CAR into the subject, wherein after the        introducing, the T cell and/or the NK cell including the nucleic        acid encoding the MRB-CAR expresses the MRB-CAR and binds to the        cell expressing the cognate antigen in the subject; and    -   b. administering a pharmacologic agent to the subject in        sufficient amount to increase blood pH and/or pH of a tissue        and/or pH of a microenvironment, wherein the administering is        performed before, during, or after the introducing, and wherein        the increased pH of the blood, the tissue, and/or the        microenvironment modulates binding of the MRB-CAR expressing T        cell and/or NK cell to the cell expressing the cognate antigen        in the blood, the tissue, or the microenvironment with the        increased pH.

In another aspect, provided herein is a method for alleviating on targetoff tumor toxicity in a subject, including:

-   -   a. introducing a nucleic acid encoding an microenvironment        restricted biologic chimeric antigen receptor (MRB-CAR) into a T        cell or NK cell of the subject to produce a T cell and/or NK        cell including a nucleic acid encoding the MRB-CAR;    -   b. introducing the T cell and/or NK cell including the nucleic        acid encoding the MRB-CAR into the subject, wherein after the        introducing, the T cell and/or the NK cell including the nucleic        acid encoding the MRB-CAR expresses the MRB-CAR and binds to the        cell expressing the cognate antigen in the subject; and    -   c. administering a pharmacologic agent to the subject in        sufficient amount to increase blood pH and/or pH of a tissue        and/or pH of a microenvironment to modulate binding of the        MRB-CAR to its cognate antigen in the blood, the tissue, and/or        the microenvironment with the increased pH, thereby alleviating        on target off tumor toxicity in the subject.

In some embodiments, the nucleic acid can be a vector. In illustrativeembodiments, the vector is a retroviral particle.

In another aspect, provided herein is a method for controlling bindingof a T cell and/or NK cell to a target mammalian cell, including:

-   -   a. contacting the target mammalian cell with the T cell and/or        NK cell in a microenvironment, wherein the target mammalian cell        expresses a cognate antigen, and the T cell and/or NK cell        expresses a microenvironment restricted biologic chimeric        antigen receptor (MRB-CAR) that binds to the cognate antigen        differentially at pH 6.7 as compared to pH 7.4; and    -   b. increasing the pH of the microenvironment by introducing a        pharmacologic agent to the microenvironment in sufficient        amount, thereby controlling the binding of the T cell and/or NK        cell to the target mammalian cell.

In another aspect, provided herein is a method for controlling thebinding of a T cell and/or NK cell expressing a microenvironmentrestricted biologic chimeric antigen receptor (MRB-CAR) to a targetmammalian cell in a subject in vivo, including administering apH-modulating pharmacologic agent to the subject through an effectivedosing regimen that increases the pH of a microenvironment within thesubject, wherein the subject includes the T cell and/or the NK cellexpressing the MRB-CAR, wherein the MRB-CAR binds to its cognate antigendifferentially at pH 6.7 as compared to pH 7.4, wherein themicroenvironment include the target mammalian cell, wherein the targetmammalian cell expresses the cognate antigen on its surface, and whereinthe T cell and/or NK cell binds to the target mammalian celldifferentially before versus after the pH of the microenvironment isincreased, thereby controlling the binding of the T cell and/or NK cellto the target mammalian cell in a subject in vivo.

In any of the aspects provided herein that include a pharmacologic agentand an MRB-CAR, the MRB-CAR can have reduced binding to its cognateantigen at one pH than at a different pH. In illustrative embodimentswhere illustrative pH values for differential binding of an MRB-CAR arenot already provided in the broadest aspect and alternatively for otherembodiments in place of those values for such aspects, the MRB-CAR canhave reduced binding at a higher pH than at a lower pH. For example, theMRB-CAR can have reduced binding to its cognate antigen at a pH above7.0, 7.1, 7.2, 7.3, 7.4, or 7.5 than at a pH below 6.4, 6.5, 6.6, 6.7,6.8, 6.9, or 7.0. In other embodiments, the MRB-CAR can have reducedbinding at a higher pH than at a lower pH. For example, the MRB-CAR canhave reduced binding to its cognate antigen at a pH below 6.4, 6.5, 6.6,6.7, 6.8, 6.9, or 7.0 than at a pH above 7.0, 7.1, 7.2, 7.3, 7.4, or7.5. In some illustrative embodiments, the MRB-CAR exhibits increasedbinding at a pH of 6.5 to 6.7 compared to pH 7.4 to 7.6. In otherillustrative embodiments, the MRB-CAR exhibits increased binding at a pHof 6.7 compared to a pH of 7.4. In other embodiments, the MRB-CARexhibits increased binding in the pH of a tumor compared to the pH ofblood. In some embodiments, the MRB-CAR can include an antigen-specifictargeting region, a stalk, and an intracellular activating domain. Insome embodiments, the MRB-CAR can also include a co-stimulatory domain.In some embodiments, the MRB-CAR can bind to a tumor associated antigen.

In any of the aspects provided herein that include a pharmacologic agentand an MRB-CAR, the pH of the microenvironment can be increased from apH below 7.0 to a pH above 7.0. For example, the pH can be increasedfrom a pH below 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0 to a pH above 7.0,7.1, 7.2, 7.3, or 7.4. In some embodiments, the MRB-CAR can bind to thecognate antigen at the increased pH but not a pH of the microenvironmentbefore introducing the pharmacologic agent. In certain embodiments, thepH can be increased from below 7.0 to a pH of 7.1 to 8.0 or to a pH of7.1 to 7.8 or to a pH of 7.2 to 7.8 or a pH of 7.2 to 7.6 or a pH of 7.3to 7.6 or to a pH of 7.4 to 7.8 or to a pH of 7.4 to 7.6. Such anincrease in pH can occur for less than 1, 2, 4, 6, 8, 12, or 24 hours orfor more than 1, 2, 4, 6, 8, 12 or 24 hours depending on the type anddose of pharmacologic agent administered. In certain embodiments, thepharmacologic agent is administered such that the pH remains above 7.0,7.1, 7.2, 7.3, 7.4, or 7.5; or between 7.0, 7.1, 7.2, 7.3 on the low endof the range and 7.4, 7.5, 7.6, 7.7, or 7.8 on the high end of therange, in the target tissue, such as a tumor, and for example in atleast a surface of a target tissue (e.g. tumor) microenvironment, in atleast a portion of a target tissue (e.g. tumor) microenvironment, and inillustrative embodiments throughout a target tissue (e.g. tumor)microenvironment.

In any of the aspects provided herein that include a pharmacologic agentand an MRB-CAR, the microenvironment can be an in vivo microenvironment,such as a tumor, a tissue, a non-tumor tissue, a normal tissue, or atissue that has undergone a transient shift in pH. For example, tissuesthat typically undergo transient shifts in pH include a muscle tissue inanaerobic conditions or muscle tissue undergoing exercise or an inflamedtissue or a tissue experiencing inflammation. In some embodiments thatinclude a target mammalian cell, the target mammalian cell can be atumor cell or a non-tumor or normal cell.

In any of the aspects provided herein that include a pharmacologic agentand an MRB-CAR, the pharmacologic agent can be sodium bicarbonate,tris-hydroxylmethyl aminomethane, an equimolar hypertonic solution ofsodium bicarbonate and sodium carbonate, or proton pump inhibitors suchesomeprazole, esomeprazole and naproxen, lansoprazole, omeprazole, andrabeprazole.

Nucleic acids encoding MRB-CARs of the present disclosure can beintroduced through various means into T cells and/or NK cells. In any ofthe aspects provided herein that include a pharmacologic agent and anMRB-CAR, the introducing step or steps can be performed by

-   -   a. contacting resting T cells and/or NK cells of the subject ex        vivo without requiring prior ex vivo stimulation, with a        replication incompetent recombinant retroviral particle        including:        -   i. one or more pseudotyping elements on its surface that is            capable of binding to a T cell and/or NK cell and            facilitating membrane fusion of the replication incompetent            recombinant retroviral particle thereto; and        -   ii. a polynucleotide including a transcriptional unit            operatively linked to a promoter active in T cells and/or NK            cells, that encodes the MRB-CAR,    -   wherein said contacting facilitates transduction of at least        some of the resting T cells and/or NK cells by the replication        incompetent recombinant retroviral particle, thereby producing T        cells and/or NK cells capable of expressing the MRB-CAR,        typically because they now include the polynucleotide that        includes a transcriptional unit operatively linked to a promoter        active in T cells and/or NK cells, that encodes the MRB-CAR; and    -   b. introducing the T cells and/or NK cells capable of expressing        the MRB-CAR into the subject.

In some embodiments, the T cells and/or NK cells can undergo 2, 3, 4, 5,6, 7, 8, 9, or 10 or fewer cells divisions ex vivo prior to beingintroduced. In some embodiments, the resting T cells and/or resting NKcells can be in contact with the replication incompetent recombinantretroviral particle for between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12hours on the low end of the range and 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours on the high end of therange, where for a given range a low value is below a high value. Insome embodiments, the resting T cells and/or resting NK cells can befrom blood which has been collected from the subject. In illustrativeembodiments, no more than 12, 15. 16, 18, 21, 24, 30, 36, 42, or 48hours can pass between the time the blood is collected from the subjectand the time the T cells and/or resting NK cells capable of expressingthe MRB-CAR are introduced into the subject. In some embodiments, allthe steps after collecting the blood and before introducing the T cellsand/or resting NK cells capable of expressing the MRB-CAR can beperformed in a closed system.

In any embodiment provided herein that includes a replicationincompetent recombinant retroviral particle in a method that includes anMRB-CAR and a pharmacologic agent, the polynucleotide that includes atranscriptional unit operatively linked to a promoter active in T cellsand/or NK cells that encodes the MRB-CAR is taken up by the T cell(s)and/or NK cell(s) such that such the cell(s) is capable of expressingthe MRB-CAR. In illustrative embodiments, the T cell(s) and/or NKcell(s) integrate the polynucleotide into their genome.

In any embodiment provided herein that includes a replicationincompetent recombinant retroviral particle in a method that includes anMRB-CAR and a pharmacologic agent, the replication incompetentrecombinant retroviral particle can further include an activationelement on its surface, such as an activation element that is capable ofactivating a resting T cell and/or resting NK cell. In some embodiments,the activation element can include any activation element provided inthis disclosure. In illustrative embodiments, the activation element caninclude a membrane-bound polypeptide capable of binding to CD3 and/or amembrane-bound polypeptide capable of binding to CD28. In any of theembodiments that includes an activation element on the surface ofreplication incompetent recombinant retroviral particle in a method thatincludes an MRB-CAR and a pharmacologic agent, one or more of themembrane-bound polypeptides can be fused to a heterologous GPI anchorattachment sequence. In some embodiments, the membrane-bound polypeptidecapable of binding CD3 and/or the membrane-bound polypeptide capable ofbinding CD28 can be an scFv or scFvFc that binds CD3 or CD28,respectively. In illustrative embodiments, the membrane-boundpolypeptide capable of binding CD3 can be an scFv or scFvFc that bindsCD3. In some embodiments, the membrane-bound polypeptide capable ofbinding CD28 can be the extracellular domains of CD80, CD86, or afunctional fragment thereof that is capable of inducing CD28-mediatedactivation of Akt.

In any embodiment provided herein that includes a replicationincompetent recombinant retroviral particle in a method that includes anMRB-CAR and a pharmacologic agent, the polynucleotide encoding theMRB-CAR can be operably linked to a riboswitch. In some embodiments, theriboswitch can be capable of binding a nucleoside analog. In someembodiments, the nucleoside analog can be an antiviral drug, such asacyclovir or penciclovir.

In any embodiment provided herein that includes a replicationincompetent recombinant retroviral particle in a method that includes anMRB-CAR and a pharmacologic agent, the replication incompetentrecombinant retroviral particle can include on its surface a recognitiondomain of a monoclonal antibody approved biologic. For example, therecognition domain can include a polypeptide that is recognized by anantibody that recognizes EGFR, or an epitope thereof.

In any embodiment provided herein that includes a replicationincompetent recombinant retroviral particle in a method that includes anMRB-CAR and a pharmacologic agent, the one or more pseudotyping elementscan include a Measles Virus F polypeptide, a Measles Virus Hpolypeptide, and/or a fragment thereof that retains the ability to bindto resting T cells and/or resting NK cells. In some embodiments, the oneor more pseudotyping elements can include a VSV-G polypeptide. In someembodiments, the replication incompetent recombinant retroviral particlecan include on its surface a fusion polypeptide of IL-7, or an activefragment thereof, and DAF including a GPI anchor attachment sequence.

In any embodiment provided herein that includes a replicationincompetent recombinant retroviral particle in a method that includes anMRB-CAR and a pharmacologic agent, the genome of the replicationincompetent recombinant retroviral particle can encode one or moreinhibitory RNA molecules, for example two or more, three or more, fouror more, five or more, or six or more inhibitory RNA molecules. In someembodiments, the inhibitory RNA molecules can be directed againstdifferent RNA targets. In some embodiments, the inhibitory RNA moleculescan be located within an intron. In some embodiments, the inhibitory RNAmolecules are capable of forming a 5′ stem and a 3′ stem that form a18-25 nucleotide RNA duplex. In some embodiments, at least one of theinhibitory RNA molecules can include from 5′ to 3′ orientation: a 5′microRNA flanking sequence, a 5′ stem, a loop, a 3′ stem, and a 3′microRNA flanking sequence, wherein the 5′ stem or the 3′ stem iscapable of binding to an RNA target. In further embodiments, the 5′ stemcan 18 to 25 nucleotides in length, wherein said 3′ stem is 18 to 25nucleotides in length, wherein said loop is 3 to 40 nucleotides inlength. In some embodiments, one or more of the 5′ microRNA flankingsequence and the 3′ microRNA flanking sequence can be derived from anaturally occurring miRNA, such as mIR-155.

In another aspect, provided herein is a pH-modulating pharmacologicagent for use in a method for controlling the binding of a T cell and/orNK cell to a target mammalian cell in a subject in vivo, includingadministering the pH-modulating pharmacologic agent to the subjectthrough an effective dosing regimen that increases the pH of amicroenvironment within the subject, wherein the subject includes the Tcell and/or the NK cell, wherein the T cell and/or NK cell expresses amicroenvironment restricted biologic chimeric antigen receptor (MRB-CAR)that binds to its cognate antigen differentially at pH 6.7 as comparedto pH 7.4, wherein the T cell and/or NK cell expresses the MRB-CAR,wherein the microenvironment includes the target mammalian cell, whereinthe target mammalian cell expresses the cognate antigen on theirsurface, and wherein the T cell and/or NK cell binds to the targetmammalian cell differentially before versus after the pH of themicroenvironment is increased by administering the pH-modulatingpharmacologic agent thereby controlling the binding of the T cell and/orNK cell to the target mammalian cell in a subject in vivo.

In another aspect, provided herein is a pharmacologic agent for use in amethod for modulating the binding of a microenvironment restrictedbiologic chimeric antigen receptor (MRB-CAR) expressing T cell or NKcell to a cell expressing a cognate antigen of the MRB-CAR in a subject,for treating tumor growth, wherein the method includes:

-   -   a. introducing a T cell and/or NK cell capable of expressing the        MRB-CAR into the subject, wherein the MRB-CAR binds to the cell        expressing the cognate antigen in the subject, wherein after the        introducing, the T cell and/or the NK cell including the nucleic        acid encoding the MRB-CAR expresses the MRB-CAR and binds to the        cell expressing the cognate antigen in the subject; and    -   b. administering the pharmacologic agent to the subject in        sufficient amount to increase blood pH and/or a tissue pH and/or        a microenvironment pH to modulate binding of the MRB-CAR        expressing T cell and/or NK cell to the cell expressing the        cognate antigen in the blood, the tissue, or the        microenvironment with the increased pH.

In another aspect, provided herein is a pharmacologic agent for use in amethod for alleviating on target off tumor toxicity in a subject,wherein the method includes:

-   -   a. introducing a nucleic acid encoding a microenvironment        restricted biologic chimeric antigen receptor (MRB-CAR) into a T        cell or NK cell of the subject, to produce a T cell and/or NK        cell capable of expressing the MRB-CAR;    -   b. introducing the T cell and/or NK cell capable of expressing        the MRB-CAR into the subject, wherein after the introducing, the        T cell and/or the NK cell including the nucleic acid encoding        the MRB-CAR expresses the MRB-CAR and binds to the cell        expressing the cognate antigen in the subject; and    -   c. administering the pharmacologic agent to the subject in        sufficient amount to increase blood pH and/or a tissue pH and/or        a microenvironment pH to modulate binding of the MRB-CAR to its        cognate antigen in the blood, the tissue, and/or the        microenvironment with the increased pH, thereby alleviating on        target off tumor toxicity in the subject.

In another aspect, provided herein is a pharmacologic agent for use in amethod for controlling the binding of a T cell and/or NK cell expressinga microenvironment restricted biologic chimeric antigen receptor(MRB-CAR) to a target mammalian cell, for treating tumor growth, whereinthe method includes:

-   -   a. contacting the target mammalian cell with the T cell and/or        NK cell expressing the MRB-CAR in a microenvironment, wherein        the target mammalian cell expresses a cognate antigen, and the T        cell and/or NK cell expresses the MRB-CAR that binds to the        cognate antigen differentially at pH 6.7 as compared to pH 7.4;        and    -   b. increasing the pH of the microenvironment by introducing the        pharmacologic agent to the microenvironment in sufficient        amount, thereby controlling the binding of the T cell and/or NK        cell expressing the MRB-CAR to the target mammalian cell.

In another aspect, provided herein is a pharmacologic agent for use in amethod for controlling the binding of a T cell and/or NK cell expressinga microenvironment restricted biologic chimeric antigen receptor(MRB-CAR) to a target mammalian cell in a subject in vivo, for treatingtumor growth, wherein the pharmacologic agent is a pH-modulatingpharmacologic agent, and wherein the method includes administering thepH-modulating pharmacologic agent to the subject through an effectivedosing regimen that increases the pH of a microenvironment within thesubject, wherein the subject includes the T cell and/or NK cellexpressing the MRB-CAR, wherein the MRB-CAR binds to its cognate antigendifferentially at pH 6.7 as compared to pH 7.4, wherein themicroenvironment includes the target mammalian cell, wherein the targetmammalian cell expresses the cognate antigen on its surface, and whereinthe T cell and/or NK cell binds to the target mammalian celldifferentially before versus after the pH of the microenvironment isincreased.

In another aspect, provided herein is a pH-modulating pharmacologicagent for use in a method for controlling the binding of a T cell and/orNK cell expressing a microenvironment restricted biologic chimericantigen receptor (MRB-CAR) to a target mammalian cell in a subject invivo, for treating tumor growth, wherein the method includesadministering the pH-modulating pharmacologic agent to the subjectthrough an effective dosing regimen that increases the pH of amicroenvironment within the subject, wherein the subject includes the Tcell and/or NK cell expressing the MRB-CAR, wherein the MRB-CAR binds toits cognate antigen differentially at pH 6.7 as compared to pH 7.4,wherein the microenvironment includes the target mammalian cell, whereinthe target mammalian cell expresses the cognate antigen on its surface,and wherein the T cell and/or NK cell binds to the target mammalian celldifferentially before versus after the pH of the microenvironment isincreased by administering the pH-modulating pharmacologic agent.

In another aspect, provided herein is a use of a pH-modulatingpharmacologic agent for use in the manufacture of a medicament forcontrolling the binding of a T cell and/or NK cell expressing amicroenvironment restricted biologic chimeric antigen receptor (MRB-CAR)to a target mammalian cell in a subject in vivo, wherein thepH-modulating pharmacologic agent is to be administered to the subjectthrough an effective dosing regimen that increases the pH of amicroenvironment within the subject, wherein the subject includes the Tcell and/or NK cell expressing the MRB-CAR, wherein the MRB-CAR binds toits cognate antigen differentially at pH 6.7 as compared to pH 7.4,wherein the microenvironment includes the target mammalian cell, whereinthe target mammalian cell expresses the cognate antigen on theirsurface, and wherein the T cell binds to the target mammalian celldifferentially before versus after the pH of the microenvironment isincreased by administering the pH-modulating pharmacologic agent.

In any of the aspects provided herein that include a pH-modulatingpharmacologic agent or a pharmacologic agent for use in a method and anMRB-CAR or include the use of a pH-modulating pharmacologic agent and anMRB-CAR, the MRB-CAR can have reduced binding to its cognate antigen atone pH than at a different pH. In illustrative embodiments whereillustrative pH values for differential binding of an MRB-CAR are notalready provided in the broadest aspect and alternatively for otherembodiments in place of those values for such aspects, the MRB-CAR canhave reduced binding at a higher pH than at a lower pH. For example, theMRB-CAR can have reduced binding to its cognate antigen at a pH above7.0, 7.1, 7.2, 7.3, 7.4, or 7.5 than at a pH below 6.4, 6.5, 6.6, 6.7,6.8, 6.9, or 7.0. In other embodiments, the MRB-CAR can have reducedbinding at a higher pH than at a lower pH. For example, the MRB-CAR canhave reduced binding to its cognate antigen at a pH below 6.4, 6.5, 6.6,6.7, 6.8, 6.9, or 7.0 than at a pH above 7.0, 7.1, 7.2, 7.3, 7.4, or7.5. In some illustrative embodiments, the MRB-CAR exhibits increasedbinding at a pH of 6.5 to 6.7 compared to pH 7.4 to 7.6. In otherillustrative embodiments, the MRB-CAR exhibits increased binding at a pHof 6.7 compared to a pH of 7.4. In other embodiments, the MRB-CARexhibits increased binding in the pH of a tumor compared to the pH ofblood. In some embodiments, the MRB-CAR can include an antigen-specifictargeting region, a stalk, and an intracellular activating domain. Insome embodiments, the MRB-CAR can also include a co-stimulatory domain.In some embodiments, the MRB-CAR can bind to a tumor associated antigen.

In any of the aspects provided herein that include a pH-modulatingpharmacologic agent or a pharmacologic agent for use in a method and anMRB-CAR or include the use of a pH-modulating pharmacologic agent and anMRB-CAR, the pH of the microenvironment can be increased from a pH below7.0 to a pH above 7.0. For example, the pH can be increased from a pHbelow 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0 to a pH above 7.0, 7.1, 7.2,7.3, or 7.4. In some embodiments, the MRB-CAR can bind to the cognateantigen at the increased pH but not a pH of the microenvironment beforeintroducing the pharmacologic agent. In certain embodiments, the pH canbe increased from below 7.0 to a pH of 7.1 to 8.0 or to a pH of 7.1 to7.8 or to a pH of 7.2 to 7.8 or a pH of 7.2 to 7.6 or a pH of 7.3 to 7.6or to a pH of 7.4 to 7.8 or to a pH of 7.4 to 7.6. Such an increase inpH can occur for less than 1, 2, 4, 6, 8, 12, or 24 hours or for morethan 1, 2, 4, 6, 8, 12 or 24 hours depending on the type and dose ofpharmacologic agent administered. In certain embodiments, thepharmacologic agent is administered such that the pH remains above 7.0,7.1, 7.2, 7.3, 7.4, or 7.5; or between 7.0, 7.1, 7.2, 7.3 on the low endof the range and 7.4, 7.5, 7.6, 7.7, or 7.8 on the high end of therange, in the target tissue, such as a tumor, and for example in atleast a surface of a target tissue (e.g. tumor) microenvironment, in atleast a portion of a target tissue (e.g. tumor) microenvironment, and inillustrative embodiments throughout a target tissue (e.g. tumor)microenvironment.

In any of the aspects provided herein that include a pH-modulatingpharmacologic agent or a pharmacologic agent for use in a method and anMRB-CAR or include the use of a pH-modulating pharmacologic agent and anMRB-CAR, the microenvironment can be an in vivo microenvironment, suchas a tumor, a tissue, a non-tumor tissue, a normal tissue, or a tissuethat has undergone a transient shift in pH. For example, tissues thattypically undergo transient shifts in pH include a muscle tissue inanaerobic conditions or muscle tissue undergoing exercise or an inflamedtissue or a tissue experiencing inflammation. In some embodiments thatinclude a target mammalian cell, the target mammalian cell can be atumor cell or a non-tumor or normal cell.

In any of the aspects provided herein that include a pH-modulatingpharmacologic agent or a pharmacologic agent for use in a method and anMRB-CAR or include the use of a pH-modulating pharmacologic agent and anMRB-CAR, the pharmacologic agent can be sodium bicarbonate,tris-hydroxylmethyl aminomethane, an equimolar hypertonic solution ofsodium bicarbonate and sodium carbonate, or proton pump inhibitors suchesomeprazole, esomeprazole and naproxen, lansoprazole, omeprazole, andrabeprazole.

In any of the aspects provided herein that include a pH-modulatingpharmacologic agent or a pharmacologic agent for use in a method and anMRB-CAR or include the use of a pH-modulating pharmacologic agent and anMRB-CAR, the pharmacologic agent can be used in a method for thetreatment of cancer, tumors, tumor growth, or a cell proliferativedisorder.

In another aspect, provided herein is a kit containing a containercontaining a replication incompetent recombinant retroviral particle,and instructions for use thereof for treating tumor growth, wherein theinstructions instruct a method for controlling the binding of a T celland/or NK cell to a target mammalian cell, in a method including:

-   -   a. transducing the T cell and/or NK cell with the replication        incompetent recombinant retroviral particle including in its        genome a microenvironment restricted biologic chimeric antigen        receptor (MRB-CAR) that binds to the cognate antigen        differentially at pH 6.7 as compared to pH 7.4 to produce a T        cell and/or NK cell capable of expressing the MRB-CAR;    -   b. introducing the T cell and/or NK cell capable of expressing        the MRB-CAR into the subject, wherein after the introducing, the        T cell and/or the NK cell including the nucleic acid encoding        the MRB-CAR expresses the MRB-CAR and binds to the cell        expressing the cognate antigen in the subject;    -   c. contacting the target mammalian cell with the MRB-CAR        expressing T cell and/or NK cell in a microenvironment, wherein        the target mammalian cell expresses a cognate antigen of the        MRB-CAR, and the T cell and/or NK cell expresses the MRB-CAR;        and    -   d. increasing the pH of the microenvironment by introducing a        pH-modulating pharmacologic agent to the microenvironment in        sufficient amount, thereby affecting the binding of the target        mammalian cell with the T cell and/or NK cell.        In some embodiments, the kit can further include a pH-modulating        pharmacologic agent.

In some embodiments of the kit, the MRB-CAR can have reduced binding toits cognate antigen at one pH than at a different pH. In illustrativeembodiments where illustrative pH values for differential binding of anMRB-CAR are not already provided in the broadest aspect andalternatively for other embodiments in place of those values for suchaspects, the MRB-CAR can have reduced binding at a higher pH than at alower pH. For example, the MRB-CAR can have reduced binding to itscognate antigen at a pH above 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5 than at apH below 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. In other embodiments, theMRB-CAR can have reduced binding at a higher pH than at a lower pH. Forexample, the MRB-CAR can have reduced binding to its cognate antigen ata pH below 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0 than at a pH above 7.0,7.1, 7.2, 7.3, 7.4, or 7.5. In some illustrative embodiments, theMRB-CAR exhibits increased binding at a pH of 6.5 to 6.7 compared to pH7.4 to 7.6. In other illustrative embodiments, the MRB-CAR exhibitsincreased binding at a pH of 6.7 compared to a pH of 7.4. In otherembodiments, the MRB-CAR exhibits increased binding in the pH of a tumorcompared to the pH of blood. In some embodiments, the MRB-CAR caninclude an antigen-specific targeting region, a stalk, and anintracellular activating domain. In some embodiments, the MRB-CAR canalso include a co-stimulatory domain. In some embodiments, the MRB-CARcan bind to a tumor associated antigen.

In some embodiments of the kit, the pH of the microenvironment can beincreased from a pH below 7.0 to a pH above 7.0. For example, the pH canbe increased from a pH below 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0 to apH above 7.0, 7.1, 7.2, 7.3, or 7.4. In some embodiments, the MRB-CARcan bind to the cognate antigen at the increased pH but not a pH of themicroenvironment before introducing the pharmacologic agent. In certainembodiments, the pH can be increased from below 7.0 to a pH of 7.1 to8.0 or to a pH of 7.1 to 7.8 or to a pH of 7.2 to 7.8 or a pH of 7.2 to7.6 or a pH of 7.3 to 7.6 or to a pH of 7.4 to 7.8 or to a pH of 7.4 to7.6. Such an increase in pH can occur for less than 1, 2, 4, 6, 8, 12,or 24 hours or for more than 1, 2, 4, 6, 8, 12 or 24 hours depending onthe type and dose of pharmacologic agent administered. In certainembodiments, the pharmacologic agent is administered such that the pHremains above 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5; or between 7.0, 7.1, 7.2,7.3 on the low end of the range and 7.4, 7.5, 7.6, 7.7, or 7.8 on thehigh end of the range, in the target tissue, such as a tumor, and forexample in at least a surface of a target tissue (e.g. tumor)microenvironment, in at least a portion of a target tissue (e.g. tumor)microenvironment, and in illustrative embodiments throughout a targettissue (e.g. tumor) microenvironment. In some embodiments, themicroenvironment can be an in vivo microenvironment, such as a tumor, atissue, a non-tumor tissue, a normal tissue, or a tissue that hasundergone a transient shift in pH. For example, tissues that typicallyundergo transient shifts in pH include a muscle tissue in anaerobicconditions or muscle tissue undergoing exercise or an inflamed tissue ora tissue experiencing inflammation. In some embodiments that include atarget mammalian cell, the target mammalian cell can be a tumor cell ora non-tumor or normal cell.

In some embodiments of the kit, the pharmacologic agent can be sodiumbicarbonate, tris-hydroxylmethyl aminomethane, an equimolar hypertonicsolution of sodium bicarbonate and sodium carbonate, or proton pumpinhibitors such esomeprazole, esomeprazole and naproxen, lansoprazole,omeprazole, and rabeprazole.

In one aspect, provided herein is a replication incompetent recombinantretroviral particle comprising in its genome a polynucleotide comprisingone or more nucleic acid sequences operatively linked to a promoteractive in T cells and/or NK cells, wherein:

-   -   a. a first nucleic acid sequence of the one or more nucleic acid        sequences encodes one or more (e.g. two or more) inhibitory RNA        molecules directed against one or more RNA targets, and b. a        second nucleic acid sequence of the one or more nucleic acid        sequences encodes a chimeric antigen receptor (CAR) comprising        an antigen-specific targeting region (ASTR), a transmembrane        domain, and an intracellular activating domain.

Provided in another aspect herein is a mammalian packaging cell linecomprising a packageable RNA genome for a replication incompetentretroviral particle, wherein said packageable RNA genome comprises:

-   -   a. a 5′ long terminal repeat, or active fragment thereof;    -   b. a nucleic acid sequence encoding a retroviral cis-acting RNA        packaging element;    -   c. a polynucleotide comprising one or more nucleic acid        sequences operatively linked to a promoter active in T cells        and/or NK cells, wherein a first nucleic acid sequence of the        one or more nucleic acids encodes one or more (e.g. two or more)        inhibitory RNA molecules directed against one or more RNA        targets and a second nucleic acid sequence of the one or more        nucleic acid sequences encodes a chimeric antigen receptor (CAR)        comprising an antigen-specific targeting region (ASTR), a        transmembrane domain, and an intracellular activating domain;        and    -   d. a 3′ long terminal repeat, or active fragment thereof.

In some embodiments of the mammalian packaging cell line aspect, thepolynucleotide of (c) can be in reverse orientation to the nucleic acidsequence encoding the retroviral cis-acting RNA packaging element (b),the 5′ long terminal repeat (a), and/or the 3′ long terminal repeat (d).

In some embodiments of the mammalian packaging cell line aspect,expression of the packageable RNA genome is driven by an induciblepromoter active in the mammalian packaging cell line.

In some embodiments of the mammalian packaging cell line aspect, theretroviral cis-acting RNA packaging element can comprise a centralpolypurine tract (cPPT)/central termination sequence, an HIV Psi, or acombination thereof.

Provided in another aspect herein is a retroviral vector comprising apackageable RNA genome for a replication incompetent retroviralparticle, wherein said packageable RNA genome comprises:

-   -   a. a 5′ long terminal repeat, or active fragment thereof;    -   b. a nucleic acid sequence encoding a retroviral cis-acting RNA        packaging element;    -   c. a polynucleotide comprising one or more nucleic acid        sequences operatively linked to a promoter active in T cells        and/or NK cells, wherein a first nucleic acid sequence of the        one or more nucleic acids encodes one or more (e.g. two or more)        inhibitory RNA molecules directed against one or more RNA        targets and a second nucleic acid sequence of the one or more        nucleic acid sequences encodes a chimeric antigen receptor (CAR)        comprising an antigen-specific targeting region (ASTR), a        transmembrane domain, and an intracellular activating domain;        and    -   d. a 3′ long terminal repeat, or active fragment thereof.

In some embodiments of the retroviral vector aspect, the polynucleotideof (c) can be in reverse orientation to the nucleic acid sequenceencoding the retroviral cis-acting RNA packaging element (b), the 5′long terminal repeat (a), and/or the 3′ long terminal repeat (d).

In some embodiments of the retroviral vector aspect, expression of thepackageable RNA genome is driven by an inducible promoter active in themammalian packaging cell line.

In some embodiments of the retroviral vector aspect, the retroviralcis-acting RNA packaging element can comprise a central polypurine tract(cPPT)/central termination sequence, an HIV Psi, or a combinationthereof. The retroviral vector can optionally include an antibioticresistance gene and/or a detectable marker.

Provided herein in another aspect is a method for genetically modifyingor transducing a lymphocyte (e.g. a T cell or an NK cell) or apopulation thereof, of a subject, comprising contacting the lymphocyte(e.g. the T cell or NK cell) or a population thereof, of the subject exvivo, with a replication incompetent recombinant retroviral particlecomprising in its genome a polynucleotide comprising one or more nucleicacid sequences operatively linked to a promoter active in lymphocytes(e.g. T cells and/or NK cells), wherein a first nucleic acid sequence ofthe one or more nucleic acid sequences encodes one or more (e.g. two ormore) inhibitory RNA molecules directed against one or more RNA targetsand a second nucleic acid sequence of the one or more nucleic acidsequences encodes a chimeric antigen receptor (CAR) comprising anantigen-specific targeting region (ASTR), a transmembrane domain, and anintracellular activating domain, wherein said contacting facilitatesgenetic modification and/or transduction of the lymphocyte (e.g. T cellor NK cell), or at least some of the lymphocytes (e.g. T cells and/or NKcells) by the replication incompetent recombinant retroviral particle,thereby producing a genetically modified and/or transduced lymphocyte(e.g. T cell and/or NK cell).

In some embodiments of the method provided immediately above, thegenetically modified and/or transduced lymphocyte (e.g. T cell and/or NKcell) or population thereof, is introduced into the subject. In someembodiments, the genetically modified and/or transduced lymphocyte (e.g.T cell and/or NK cell) or population thereof, undergoes 4 or fewer celldivisions ex vivo prior to being introduced or reintroduced into thesubject. In some embodiments, the lymphocyte(s) are resting T cellsand/or resting NK cells that are in contact with the replicationincompetent recombinant retroviral particles for between 1 hour and 12hours. In some embodiments, no more than 8 hours pass between the timeblood is collected from the subject and the time the geneticallymodified T cells and/or NK cells are reintroduced into the subject. Insome embodiments, all steps after the blood is collected and before theblood is reintroduced, are performed in a closed system in which aperson monitors the closed system throughout the processing.

Provided herein in another aspect is a genetically modified T celland/or NK cell comprising:

-   -   a. one or more (e.g. two or more) inhibitory RNA molecules        directed against one or more RNA targets; and    -   b. a chimeric antigen receptor (CAR) comprising an        antigen-specific targeting region (ASTR), a transmembrane        domain, and an intracellular activating domain, wherein said one        or more (e.g. two or more) inhibitory RNA molecules and the CAR        are encoded by nucleic acid sequences that are genetic        modifications of the T cell and/or NK cell.

In some embodiments of the genetically modified T cell and/or NK cellaspect, the genetically modified T cell and/or NK cell also comprises atleast one lymphoproliferative element that is not an inhibitory RNAmolecule, wherein said lymphoproliferative element is encoded by anucleic acid that is a genetic modification of the T cell and/or NKcell. In some embodiments, the inhibitory RNA molecules, the CAR, and/orthe at least one lymphoproliferative element are expressed in apolycistronic matter. In illustrative embodiments, the inhibitory RNAmolecules are expressed from a single polycistronic transcript.

Provided herein in another aspect is a replication incompetentrecombinant retroviral particle for use in a method for geneticallymodifying a lymphocyte of a subject, for treating tumor growth, whereinthe replication incompetent recombinant retroviral particle comprises inits genome a polynucleotide comprising one or more nucleic acidsequences operatively linked to a promoter active in T cells and/or NKcells, wherein a first nucleic acid sequence of the one or more nucleicacid sequences encodes one or more (e.g. two or more) inhibitory RNAmolecules directed against one or more RNA targets and a second nucleicacid sequence of the one or more nucleic acid sequences encodes achimeric antigen receptor (CAR) comprising an antigen-specific targetingregion (ASTR), a transmembrane domain, and an intracellular activatingdomain, wherein the method comprises contacting a T cell and/or NK cellof the subject ex vivo, and said contacting facilitates transduction ofat least some of the resting T cells and/or NK cells by the replicationincompetent recombinant retroviral particles, thereby producing agenetically modified T cell and/or NK cell.

In the method for genetically modifying a lymphocyte of a subject aspectprovided immediately above, in some embodiments, a pharmacologic agentis used in the method, which further includes introducing thegenetically engineered T cell and/or an NK cell into the subject.

Provided herein in another aspect is a replication incompetentrecombinant retroviral particle for use in a method for geneticallymodifying a T cell and/or NK cell of a subject, for treating tumorgrowth, wherein the method comprises:

-   -   a. contacting the T cell and/or NK cell of the subject ex vivo,        with a replication incompetent recombinant retroviral particle        comprising in its genome a polynucleotide comprising one or more        nucleic acid sequences operatively linked to a promoter active        in T cells and/or NK cells, wherein a first nucleic acid        sequence of the one or more nucleic acid sequences encodes one        or more (e.g. two or more) inhibitory RNA molecules directed        against one or more RNA targets and a second nucleic acid        sequence of the one or more nucleic acid sequences encodes a        chimeric antigen receptor (CAR) comprising an antigen-specific        targeting region (ASTR), a transmembrane domain, and an        intracellular activating domain, wherein said contacting        facilitates transduction of at least some of the resting T cells        and/or NK cells by the replication incompetent recombinant        retroviral particles, thereby producing a genetically modified T        cell and/or NK cell; and    -   b. introducing the genetically modified T cell and/or NK cell        into the subject, thereby genetically modifying the T cell        and/or NK cell of the subject.

In the aspect provided immediately above, in some embodiments, apopulation of T cells and/or NK cells are contacted in the contactingstep, and introduced into the subject in the introducing step.

Provided herein in another aspect is the use of a replicationincompetent recombinant retroviral particle in the manufacture of a kitfor genetically modifying a T cell and/or NK cell of a subject, whereinthe use of the kit comprises:

-   -   1. contacting the T cell and/or NK cell of the subject ex vivo,        with a replication incompetent recombinant retroviral particle        comprising in its genome a polynucleotide comprising one or more        nucleic acid sequences operatively linked to a promoter active        in T cells and/or NK cells, wherein a first nucleic acid        sequence of the one or more nucleic acid sequences encodes one        or more (e.g. two or more) inhibitory RNA molecules directed        against one or more target and a second nucleic acid sequence of        the one or more nucleic acid sequences encodes a chimeric        antigen receptor (CAR) comprising an antigen-specific targeting        region (ASTR), a transmembrane domain, and an intracellular        activating domain, wherein said contacting facilitates        transduction of at least some of the resting T cells and/or NK        cells by the replication incompetent recombinant retroviral        particles, thereby producing a genetically modified T cell        and/or NK cell; and    -   2. introducing the genetically modified T cell and/or NK cell        into the subject, thereby genetically modifying the T cell        and/or NK cell of the subject.

Provided herein in another aspect is the use of a replicationincompetent recombinant retroviral particle in the manufacture of amedicament for genetically modifying a T cell and/or NK cell of asubject, wherein the use of the medicament comprises:

-   -   A) contacting the T cell and/or NK cell of the subject ex vivo,        with a replication incompetent recombinant retroviral particle        comprising in its genome a polynucleotide comprising one or more        nucleic acid sequences operatively linked to a promoter active        in T cells and/or NK cells, wherein a first nucleic acid        sequence of the one or more nucleic acid sequences encodes one        or more (e.g. two or more) inhibitory RNA molecules directed        against one or more target and a second nucleic acid sequence of        the one or more nucleic acid sequences encodes a chimeric        antigen receptor (CAR) comprising an antigen-specific targeting        region (ASTR), a transmembrane domain, and an intracellular        activating domain, wherein said contacting facilitates        transduction of at least some of the resting T cells and/or NK        cells by the replication incompetent recombinant retroviral        particles, thereby producing a genetically modified T cell        and/or NK cell; and    -   B) introducing the genetically modified T cell and/or NK cell        into the subject, thereby genetically modifying the T cell        and/or NK cell of the subject.

Provided herein in another aspect is a commercial container containing areplication incompetent recombinant retroviral particle and instructionsfor the use thereof to treat tumor growth in a subject, wherein thereplication incompetent recombinant retroviral particle comprises in itsgenome a polynucleotide comprising one or more nucleic acid sequencesoperatively linked to a promoter active in T cells and/or NK cells,wherein a first nucleic acid sequence of the one or more nucleic acidsequences encodes one or more (e.g. two or more) inhibitory RNAmolecules directed against one or more RNA targets and a second nucleicacid sequence of the one or more nucleic acid sequences encodes achimeric antigen receptor (CAR) comprising an antigen-specific targetingregion (ASTR), a transmembrane domain, and an intracellular activatingdomain.

In some embodiments, in the aspects of the commercial container, theinstructions instruct a user to contact a T cell and/or NK cell of thesubject ex vivo, to facilitate transduction of at least one resting Tcell and/or NK cell of the subject by the replication incompetentrecombinant retroviral particles, thereby producing a geneticallymodified T cell and/or NK cell.

In any of the aspects provided herein that include a polynucleotidecomprising one or more nucleic acid sequences operatively linked to apromoter active in T cells and/or NK cells, wherein a first nucleic acidsequence of the one or more nucleic acid sequences encodes one or more(e.g. two or more) inhibitory RNA molecules directed against one or moreRNA targets, and a second nucleic acid sequence of the one or morenucleic acid sequences encodes a chimeric antigen receptor (CAR)comprising an antigen-specific targeting region (ASTR), a transmembranedomain, and an intracellular activating domain, the polynucleotide mayfurther include a third nucleic acid sequence that encodes at least onelymphoproliferative element that is not an inhibitory RNA molecule. Insome embodiments, the lymphoproliferative element can be a cytokine orcytokine receptor polypeptide, or a fragment thereof comprising asignaling domain. In some embodiments, the lymphoproliferative elementis constitutively active. In certain embodiments, thelymphoproliferative element can be an IL-7 receptor or a fragmentthereof. In illustrative embodiments, the lymphoproliferative elementcan be a constitutively active IL-7 receptor or a constitutively activefragment thereof.

In any of the aspects provided herein that include a polynucleotidecomprising one or more nucleic acid sequences operatively linked to apromoter active in T cells and/or NK cells, wherein a first nucleic acidsequence of the one or more nucleic acid sequences encodes one or more(e.g. two or more) inhibitory RNA molecules directed against one or moreRNA targets, an inhibitory RNA molecule can in some embodiments includea 5′ strand and a 3′ strand that are partially or fully complementary toone another, wherein said 5′ strand and said 3′ strand are capable offorming an 18-25 nucleotide RNA duplex. In some embodiments, the 5′strand can be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length,and the 3′ strand can be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotidesin length. In some embodiments, the 5′ strand and the 3′ strand can bethe same or different lengths. In some embodiments, the RNA duplex caninclude one or more mismatches. In alternate embodiments, the RNA duplexhas no mismatches.

In any of the aspects provided herein that include a polynucleotidecomprising one or more nucleic acid sequences operatively linked to apromoter active in T cells and/or NK cells, wherein a first nucleic acidsequence of the one or more nucleic acid sequences encodes one or more(e.g. two or more) inhibitory RNA molecules directed against one or moreRNA targets, an inhibitory RNA molecule can be a miRNA or an shRNA. Insome embodiments, the inhibitory molecule can be a precursor of a miRNA,such as for example, a Pri-miRNA or a Pre-miRNA, or a precursor of anshRNA. In some embodiments, the inhibitory molecule can be anartificially derived miRNA or shRNA. In other embodiments, theinhibitory RNA molecule can be a dsRNA (either transcribed orartificially introduced) that is processed into an siRNA or the siRNAitself. In some embodiments, the inhibitory RNA molecule can be a miRNAor shRNA that has a sequence that is not found in nature, or has atleast one functional segment that is not found in nature, or has acombination of functional segments that are not found in nature. Inillustrative embodiments, at least one or all of the inhibitory RNAmolecules are miR-155.

In any of the aspects provided herein that include a polynucleotidecomprising one or more nucleic acid sequences operatively linked to apromoter active in T cells and/or NK cells, wherein a first nucleic acidsequence of the one or more nucleic acid sequences encodes one or more(e.g. two or more) inhibitory RNA molecules directed against one or moreRNA targets, an inhibitory RNA molecule, in some embodiments, cancomprises from 5′ to 3′ orientation: a 5′ arm, a 5′ stem, a loop, a 3′stem that is partially or fully complementary to said 5′ stem, and a 3′arm. In some embodiments, at least one of the two or more inhibitory RNAmolecules has this arrangement. In other embodiments, all of the two ormore inhibitory RNA molecules have this arrangement. In someembodiments, the 5′ stem can be 18, 19, 20, 21, 22, 23, 24 or 25nucleotides in length. In some embodiments, the 3′ stem can be 18, 19,20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments,the loop can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, or 40 nucleotides in length. In some embodiments, the 5′arm, 3′ arm, or both, are derived from a naturally occurring miRNA. Insome embodiments, the 5′ arm, 3′ arm, or both, are derived from anaturally occurring miRNA is selected from the group consisting of:miR-155, miR-30, miR-17-92, miR-122, and miR-21. In illustrativeembodiments, the 5′ arm, 3′ arm, or both are derived from miR-155. Insome embodiments, the 5′ arm, 3′ arm, or both are derived from Musmusculus miR-155 or Homo sapiens miR-155. In some embodiments, the 5′arm has the sequence set forth in SEQ ID NO:256 or is a functionalvariant thereof, such as, for example, a sequence that is the samelength as SEQ ID NO:256, or 95%, 90%, 85%, 80%, 75%, or 50% as long asSEQ ID NO: 256 or is 100 nucleotides or less, 95 nucleotides or less, 90nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75nucleotides or less, 70 nucleotides or less, 65 nucleotides or less, 60nucleotides or less, 55 nucleotides or less, 50 nucleotides or less, 45nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30nucleotides or less, or 25 nucleotides or less; and is at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ IDNO:256. In some embodiments, the 3′ arm has the sequence set forth inSEQ ID NO:260 or is a functional variant thereof, such as, for example,the same length as SEQ ID NO:260, or 95%, 90%, 85%, 80%, 75%, or 50% aslong as SEQ ID NO: 260 or is a sequence that is 100 nucleotides or less,95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less,80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less,65 nucleotides or less, 60 nucleotides or less, 55 nucleotides or less,50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less,35 nucleotides or less, 30 nucleotides or less, or 25 nucleotides orless; and is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or95% identical to SEQ ID NO:260. In some embodiments, the 3′ armcomprises nucleotides 221-283 of the Mus musculus BIC.

In any of the aspects provided herein that include a polynucleotidecomprising one or more nucleic acid sequences operatively linked to apromoter active in T cells and/or NK cells, wherein a first nucleic acidsequence of the one or more nucleic acid sequences encodes two or moreinhibitory RNA molecules directed against one or more RNA targets, thetwo or more inhibitory RNA molecules, in some embodiments, can bepositioned in the first nucleic acid sequence in series. In someembodiments, the inhibitory RNA molecules can be adjoined to one anothereither directly or indirectly by non-functional linker sequence(s). Insome embodiments, the linker sequences can be between 5 and 120nucleotides in length, or between 10 and 40 nucleotides in length.

In any of the aspects provided herein that include a polynucleotidecomprising one or more nucleic acid sequences operatively linked to apromoter active in T cells and/or NK cells, wherein a first nucleic acidsequence of the one or more nucleic acid sequences encodes two or moreinhibitory RNA molecules directed against one or more RNA targets, insome embodiments, the first nucleic acid sequence encodes two to fourinhibitory RNA molecules. In illustrative embodiments, between 2 and 10,2 and 8, 2 and 6, 2 and 5, 2 and 4, 3 and 5, or 3 and 6 inhibitory RNAmolecules are included in the first nucleic acid sequence. In anillustrative embodiment, four inhibitory RNA molecules are included inthe first nucleic acid sequence.

In any of the aspects provided herein that include a polynucleotidecomprising one or more nucleic acid sequences operatively linked to apromoter active in T cells and/or NK cells, wherein a first nucleic acidsequence of the one or more nucleic acid sequences encodes one or more(e.g. two or more) inhibitory RNA molecules directed against one or moreRNA targets, the one or more (e.g. two or more) inhibitory RNA moleculescan be in an intron. In some embodiments, the intron is in a promoter.In illustrative embodiments, the intron is EF-1alpha intron A. In someembodiments, the intron is adjacent to and downstream of a promoter,which in illustrative embodiments, is inactive in a packaging cell usedto produce the replication incompetent recombinant retroviral particle.

In any of the aspects provided herein that include a polynucleotidecomprising one or more nucleic acid sequences operatively linked to apromoter active in T cells and/or NK cells, wherein a first nucleic acidsequence of the one or more nucleic acid sequences encodes two or moreinhibitory RNA molecules directed against one or more RNA targets, thetwo or more inhibitory RNA molecules, in some embodiments, can bedirected against different targets. In an alternate embodiment, the twoor more inhibitory RNA molecules are directed against the same target.In some embodiments, the RNA targets are mRNAs transcribed from genesthat are expressed by T cells such as but not limited to PD-1 (preventinactivation); CTLA4 (prevent inactivation); TCRa (safety—preventautoimmunity); TCRb (safety—prevent autoimmunity); CD3Z (safety—preventautoimmunity); SOCS1 (prevent inactivation); SMAD2 (preventinactivation); a miR-155 target (promote activation); IFN gamma (reduceCRS); cCBL (prolong signaling); TRAIL2 (prevent death); PP2A (prolongsignaling); ABCG1 (increase cholesterol microdomain content by limitingclearance of cholesterol). In some embodiments, the RNA targets aremRNAs transcribed from genes that encode components of the T cellreceptor (TCR) complex. In some embodiments, at least one of the two ormore of inhibitory RNA molecules can decrease expression of T cellreceptors, in illustrative embodiments, one or more endogenous T cellreceptor(s) of a T cell. In certain embodiments, the RNA target can bemRNA transcribed from the endogenous TCRα or TCRβ gene of the T cellwhose genome comprises the first nucleic acid sequence encoding the oneor more miRNAs. In illustrative embodiments, the RNA target is mRNAtranscribed from the TCRα gene.

In any of the aspects provided herein that include a polynucleotidecomprising one or more nucleic acid sequences operatively linked to apromoter active in T cells and/or NK cells, wherein a first nucleic acidsequence of the one or more nucleic acid sequences encodes one or more(e.g. two or more) inhibitory RNA molecules directed against one or moreRNA targets, and a second nucleic acid sequence of the one or morenucleic acid sequences encodes a chimeric antigen receptor (CAR)comprising an antigen-specific targeting region (ASTR), a transmembranedomain, and an intracellular activating domain, in some embodiments, theCAR is a microenvironment restricted biologic (MRB)-CAR. In otherembodiments, the ASTR of the CAR binds to a tumor associated antigen. Inother embodiments, the ASTR of the CAR is a microenvironment-restrictedbiologic (MRB)-ASTR.

In any of the aspects provided herein that include a polynucleotidecomprising one or more nucleic acid sequences operatively linked to apromoter active in T cells and/or NK cells, wherein a first nucleic acidsequence of the one or more nucleic acid sequences encodes one or more(e.g. two or more) inhibitory RNA molecules directed against one or moreRNA targets, and a second nucleic acid sequence of the one or morenucleic acid sequences encodes a chimeric antigen receptor (CAR)comprising an antigen-specific targeting region (ASTR), a transmembranedomain, and an intracellular activating domain, and in some instances athird nucleic acid sequence of the one or more nucleic acid sequencesthat encodes at least one lymphoproliferative element that is not aninhibitory RNA molecule, in some embodiments, any or all of the firstnucleic acid sequence, second nucleic acid sequence, and third nucleicacid sequence is operably linked to a riboswitch. In some embodiments,the riboswitch is capable of binding a nucleoside analog. In someembodiments, the nucleoside analog is an antiviral drug.

In any of the aspects provided herein that include a replicationincompetent recombinant retroviral particle, in some embodiments, thereplication incompetent recombinant retroviral particle comprises apseudotyping element on its surface that is capable of binding to a Tcell and/or NK cell and facilitating membrane fusion of the replicationincompetent recombinant retroviral particle thereto. In someembodiments, the pseudotyping element can be a Measles Virus Fpolypeptide, a Measles Virus H polypeptide, a VSV-G polypeptide, or afragment of any thereof that retains the ability to bind to resting Tcells and/or resting NK cells. In illustrative embodiments, thepseudotyping element is VSV-G.

In any of the aspects provided herein that include a replicationincompetent recombinant retroviral particle, in some embodiments, thereplication incompetent recombinant retroviral particle comprises anactivation element on its surface that comprises a membrane-boundpolypeptide capable of binding to CD3; and/or a membrane-boundpolypeptide capable of binding to CD28. In some embodiments, themembrane-bound polypeptide capable of binding to CD3 is fused to aheterologous GPI anchor attachment sequence and/or the membrane-boundpolypeptide capable of binding to CD28 is fused to a heterologous GPIanchor attachment sequence. In some embodiments, the membrane-boundpolypeptide capable of binding CD3 is an anti-CD3 scFV or anti-CD3scFvFc. In illustrative embodiments, the membrane-bound polypeptidecapable of binding to CD3 is anti-CD3 scFvFc. In illustrativeembodiments, the membrane-bound polypeptide capable of binding to CD28is CD80, or an extra-cellular domain thereof, bound to a CD16B GPIanchor attachment sequence.

In any of the aspects provided herein that include a replicationincompetent recombinant retroviral particle, in some embodiments, thereplication incompetent recombinant retroviral particle comprises on itssurface a nucleic acid encoding a domain recognized by a monoclonalantibody approved biologic.

Chimeric polypeptides in the aspects provided hereinbelow that include achimeric polypeptide that promote cell proliferation of PBMCs, forexample B cells, T cells, and/or NK cells, can be referred to herein aschimeric lymphoproliferative elements (CLEs). Embodiments providedhereinbelow that include nucleic acid sequences that encode a CLE and aCAR are referred to herein as CLE CAR polynucleotide embodiments.

In embodiments and subembodiments of any of the aspects and embodimentsprovided herein that include a lymphoproliferative element, includingthose in the paragraphs above in this section, the lymphoproliferativeelement can be any of the chimeric lymphoproliferative elements providedherein, including hereinbelow in this section. In other in embodimentsand subembodiments of any of the aspects and embodiments herein thatinclude a CAR, the lymphoproliferative element in certain illustrativeembodiments is any of the chimeric lymphoproliferative elements encodedby nucleic acids in CLE CAR polynucleotide (and associated polypeptide)embodiments provided herein, for example as set out hereinbelow in thissection.

In one aspect, provided herein is an isolated polynucleotide comprisingone or more nucleic acid sequences, wherein:

-   -   a first nucleic acid sequence of the one or more nucleic acid        sequences encodes a chimeric polypeptide comprising in amino to        carboxy orientation,    -   a) an extracellular and transmembrane domain from a cytokine        receptor or a hormone receptor, wherein at least one of the        extracellular domain and the transmembrane domain comprise        -   a. a mutation that is found on a constitutively active            mutant of the cytokine receptor and wherein the            extracellular sequence does not bind a ligand of the            cytokine receptor, or        -   b. an extracellular domain and a transmembrane domain from            LMP1; and    -   b) a first intracellular domain selected from an intracellular        domain of a gene having a first intracellular domain of a        selected polypeptide identified in Tables 7-10, wherein said        chimeric polypeptide promotes cell proliferation of B cells, T        cells, and/or NK cells.

In one embodiment of the aspect in the paragraph immediately above, thefirst nucleic acid sequence further encodes a second intracellulardomain, wherein the second intracellular domain can be upstream (i.e.5′) from the first intracellular domain, or in illustrative embodimentsthe second intracellular domain is downstream from the firstintracellular domain. In a subembodiment of this embodiment, the firstintracellular domain and the second intracellular domain combinationcomprise the first intracellular domain and the second intracellulardomain combination of the selected polypeptide. Accordingly, for thesake of clarification, in a non-limiting exemplary embodiment, theselected polypeptide is M008-S212-S075. In such embodiment, the firstintracellular domain of the chimeric polypeptide comprises or is TNFRSF8transcript variant 1 (NM_001243_4) (S212) and the second polypeptidecomprises or is FCGR2C (NM_201563_5) (S075).

In one embodiment of this aspect, the chimeric polypeptide comprises thetransmembrane domain, the first intracellular domain, and the secondintracellular domain of the selected polypeptide. In one embodiment ofthis aspect, the chimeric polypeptide comprises the extracellular andtransmembrane domain, the first intracellular domain, and the secondintracellular domain of the selected polypeptide. Accordingly, for thesake of clarification, in a non-limiting exemplary embodiment, theselected polypeptide is M008-S212-S075. In such embodiment, theextracellular and transmembrane domain of the chimeric polypeptidecomprises or is Myc LMP1 (NC_007605_1) (M008), the first intracellulardomain of the chimeric polypeptide comprises or is TNFRSF8 transcriptvariant 1 (NM_001243_4) (S212), and the second intracellular domain ofthe chimeric polypeptide comprises or is FCGR2C (NM_201563_5) (S075). Ina related subembodiment, the chimeric polypeptide comprises theextracellular and transmembrane domain, the first intracellular domain,and the second intracellular domain of the selected polypeptide exceptthat the extracellular and transmembrane domain optionally comprises arecognition or elimination domain, or does not comprise a recognition orelimination domain. In a related subembodiment, the chimeric polypeptidecomprises the extracellular and transmembrane domain, the firstintracellular domain, and the second intracellular domain of theselected polypeptide except that the extracellular and transmembranedomain comprises a recognition or elimination domain of the selectedpolypeptide, or another clearance tag. In another embodiment of theaspect provided in the paragraph above, the first intracellular domainand the second intracellular domain combination comprises a firstintracellular domain of the selected polypeptide with any intracellulardomain of any gene identified in the second intracellular domains of anycandidate polypeptide in Tables 7-10.

In another embodiment of this aspect, the polynucleotide furthercomprises a second nucleic acid sequence that encodes a chimeric antigenreceptor (CAR) comprising an antigen-specific targeting region (ASTR), atransmembrane domain, and an intracellular activating domain. In asubembodiment of this aspect, the first intracellular domain and thesecond intracellular domain combination are the first intracellulardomain and the second intracellular domain combination of the selectedpolypeptide, and the selected polypeptide is identified in Tables 7-10.In another subembodiment of this embodiment, the chimeric polypeptidecomprises the extracellular and transmembrane domain, the firstintracellular domain, and the second intracellular domain of theselected polypeptide, and the selected polypeptide is identified inTables 7-10. In a related subembodiment, the chimeric polypeptidecomprises the extracellular and transmembrane domain, the firstintracellular domain, and the second intracellular domain of theselected polypeptide except that the extracellular and transmembranedomain optionally comprises a recognition or elimination domain, andwherein the recognition or elimination domain is a recognition orelimination domain of the extracellular domain of the selectedpolypeptide, or another recognition or elimination domain, and whereinthe selected polypeptide is identified in Tables 7-10.

In another aspect, provided herein is an isolated polynucleotidecomprising one or more nucleic acid sequences, wherein:

-   -   a first nucleic acid sequence of the one or more nucleic acid        sequences encodes a chimeric polypeptide comprising in amino to        carboxy orientation,    -   a) an extracellular and transmembrane domain from a cytokine        receptor or a hormone, wherein at least one of the extracellular        domain and the transmembrane domain comprise        -   c. a mutation that is found on a constitutively active            mutant of the cytokine receptor and wherein the            extracellular sequence does not bind a ligand of the            cytokine receptor, or        -   d. an extracellular domain and a transmembrane domain from            LMP1; and    -   b) a first intracellular domain selected from an intracellular        domain of CD3D, CD3E, CD8A, CD27, CD40, CD79B, IFNAR1, IL2RA,        IL3RA, IL13RA2, TNFRSF8, TNFRSF9, IL31RA, or MyD88, wherein said        chimeric polypeptide promotes cell proliferation of PBMCs, and        in illustrative embodiments, of B cells, T cells, and/or NK        cells.

In one embodiment of the aspect in the paragraph immediately above, thefirst nucleic acid sequence further encodes a second intracellulardomain, wherein the second intracellular domain can be upstream (i.e.5′) from the first intracellular domain, or in illustrative embodimentsthe second intracellular domain is downstream from the firstintracellular domain. In a subembodiment of this embodiment, the secondintracellular domain is from a gene of a second intracellular domain ina selected polypeptide identified in any table of Example 17 as yieldingan enrichment at day 35 versus day 7 above 0, 1, or 2, wherein the firstintracellular domain is from the gene of the first intracellular domainof the selected polypeptide. In a further subembodiment of thissubembodiment or embodiment, the first intracellular domain and thesecond intracellular domain combination are the first intracellulardomain and the second intracellular domain from a selected chimericpolypeptide identified in any table of Example 17 as yielding anenrichment at day 35 versus day 7 above 0, 1, or 2. In anothersubembodiment of the embodiment or subembodiments in this paragraph, thefirst intracellular domain is from a first gene of a selected firstintracellular domain of a selected polypeptide in Tables 7-10 and thesecond intracellular domain is from a second gene of a selected secondintracellular domain of the selected polypeptide. In anothersubembodiment of the embodiment or subembodiments in this paragraph, thefirst intracellular domain is from a selected polypeptide in Tables 7-10and the second intracellular domain is from the selected polypeptide inTables 7-10.

In another subembodiment of the immediately above embodiments whereinthe first nucleic acid sequence further encodes a second intracellulardomain, the second intracellular domain is selected from a secondintracellular domain identified in any of the tables of Example 17 asyielding an enrichment at day 35 versus day 7 above 0, 1, or 2, for thecorresponding first intracellular domain. In subembodiments of theseembodiments, the first intracellular domain is selected from anintracellular domain of CD27, CD40, or CD79B. In other subembodiments ofthis embodiment, the first intracellular domain is selected from anintracellular domain of CD3D, CD3E, CD8A, CD27, CD40, CD79B, IFNAR1,IL2RA, IL3RA, IL13RA2, TNFRSF8, TNFRSF9, IL31RA, or MyD88 identified inany of the tables of Example 17 as yielding an enrichment at day 35versus day 7 above 0, 1, or 2. In another subembodiment of theembodiment or subembodiments in this paragraph, the first intracellulardomain is from a first gene of a selected chimeric polypeptide in Tables7-10 and the second intracellular domain is from a second gene of theselected chimeric polypeptide in Tables 7-10. In another subembodimentof the embodiment or subembodiments in this paragraph, the firstintracellular domain is from a first gene of a selected chimericpolypeptide in Tables 7-10 and the second intracellular domain is froman intracellular domain of CD3D, CD3G, CD8A, CD8B, CD27, CD40, CD79B,CRLF2, FCGR2C, ICOS, IL2RA, IL13RA1, IL13RA2, IL15RA, TNFRSF9, andTNFRSF18. In another subembodiment of the embodiment or subembodimentsin this paragraph, the first intracellular domain is from a selectedpolypeptide identified in Tables 7-10 and the second intracellulardomain is from the selected polypeptide identified in Tables 7-10. In arelated subembodiment, the chimeric polypeptide comprises theextracellular and transmembrane domain, the first intracellular domain,and the second intracellular domain of the selected polypeptide exceptthat the extracellular and transmembrane domain optionally comprises aclearance tag, or does not comprise a clearance tag, and wherein theselected polypeptide is identified in Tables 7-10. In a relatedsubembodiment, the chimeric polypeptide comprises the extracellular andtransmembrane domain, the first intracellular domain, and the secondintracellular domain of the selected polypeptide except that theextracellular and transmembrane domain comprises a recognition orelimination domain of the selected polypeptide, or another recognitionor elimination domain, and wherein the selected polypeptide isidentified in Tables 7-10.

In another embodiment of this aspect, the first intracellular domain isselected from an intracellular domain of CD3D, CD3E, CD8A, CD27, CD40,CD79B, IFNAR1, IL2RA, IL3RA, IL13RA2, TNFRSF8, or TNFRSF9, thepolynucleotide further comprises a second nucleic acid sequence thatencodes a chimeric antigen receptor (CAR) comprising an antigen-specifictargeting region (ASTR), a transmembrane domain, and an intracellularactivating domain. In a subembodiment of this aspect, the firstintracellular domain is from a gene of the first intracellular domain ofthe selected polypeptide and the second intracellular domain is from agene of the second intracellular domain of the selected polypeptide, andthe selected polypeptide is identified in Tables 7-10. In asubembodiment of this aspect, the first intracellular domain and thesecond intracellular domain combination are the first intracellulardomain and the second intracellular domain combination of the selectedpolypeptide, and the selected polypeptide is identified in Tables 7-10.In another subembodiment of this embodiment, the chimeric polypeptidecomprises the extracellular and transmembrane domain, the firstintracellular domain, and the second intracellular domain of theselected polypeptide, and the selected polypeptide is identified inTables 7-10. In a related subembodiment, the chimeric polypeptidecomprises the extracellular and transmembrane domain, the firstintracellular domain, and the second intracellular domain of theselected polypeptide except that the extracellular and transmembranedomain optionally comprises a recognition or elimination domain, andwherein the recognition or elimination domain is a recognition orelimination domain of the extracellular domain of the selectedpolypeptide, or another recognition or elimination domain, and whereinthe selected polypeptide is identified in Tables 7-10.

In another aspect, provided herein is an isolated polynucleotidecomprising one or more nucleic acid sequences, wherein:

-   -   a first nucleic acid sequence of the one or more nucleic acid        sequences encodes a chimeric polypeptide comprising in amino to        carboxy orientation,    -   (a) an extracellular and transmembrane domain from a cytokine        receptor or a hormone, wherein at least one of the extracellular        domain and the transmembrane domain comprise        -   i. a mutation that is found on a constitutively active            mutant of the cytokine receptor and wherein the            extracellular sequence does not bind a ligand of the            cytokine receptor, or        -   ii. an extracellular domain and a transmembrane domain from            LMP1; and    -   (b) a first intracellular domain selected from an intracellular        domain of CD3D, CD3G, CD8A, CD8B, CD27, CD40, CD79B, CRLF2,        FCGR2C, ICOS, IL2RA, IL13RA1, IL13RA2, IL15RA, TNFRSF9, and        TNFRSF18, wherein said chimeric polypeptide promotes cell        proliferation of PBMCs, and in illustrative embodiments, of B        cells, T cells, and/or NK cells.

In Example 17, the intracellular domains recited in the immediatelyabove aspect were identified as noteworthy second intracellular domains.However, it will be understood that in some embodiments, intracellulardomains identified as noteworthy second intracellular domains are firstintracellular domains of the chimeric polypeptides of these embodiments.

In another aspect, provided herein is an isolated polynucleotidecomprising one or more nucleic acid sequences, wherein:

-   -   a first nucleic acid sequence of the one or more nucleic acid        sequences encodes a chimeric polypeptide comprising in amino to        carboxy orientation,    -   a) an extracellular and transmembrane domain from a cytokine        receptor or a hormone, wherein at least one of the extracellular        domain and the transmembrane domain comprise        -   a. a mutation that is found on a constitutively active            mutant of the cytokine receptor and wherein the            extracellular sequence does not bind a ligand of the            cytokine receptor, or        -   b. an extracellular domain and a transmembrane domain from            LMP1; and    -   b) a first intracellular domain selected from an intracellular        domain of CD3D, CD3E, CD8A, CD27, CD40, CD79B, IFNAR1, IL2RA,        IL3RA, IL13RA2, TNFRSF8, TNFRSF9, IL31RA, or MyD88, wherein said        chimeric polypeptide promotes cell proliferation of PBMCs, and        in illustrative embodiments, of B cells, T cells, and/or NK        cells, and wherein the extracellular and transmembrane domain is        selected from the extracellular domain and transmembrane domain        of IL7RA Ins PPCL, LMP1, CRLF2 F232C, CSF2RB V449E, CSF3R T640N,        EPOR L251C I252C, GHR E260C I270C, IL27RA F523C, and MPL S505N.

In certain embodiments of any of the above aspects directed to apolynucleotide encoding a chimeric polypeptide wherein said chimericpolypeptide promotes cell proliferation of PBMCs, and in illustrativeembodiments, of B cells, T cells, and/or NK cells, the extracellulardomain and transmembrane domain are selected from CSF3R T640N and MPLS505N. In illustrative embodiments, the extracellular domain andtransmembrane domain are from CSF3R T640N and in certain subembodiments,the isolated polynucleotide does not comprise a CAR. In certainsubembodiments, the extracellular domain and transmembrane domain areCSF3R T640N provided in Table 6 except that the recognition andelimination domain is optional. In certain embodiments, the firstintracellular domain and the second intracellular domain are from any ofthe polypeptides provided in Example 17, and the tables therein, thatinclude a recited intracellular and extracellular domain and a recitedfirst intracellular domain. In other subembodiments, the extracellularand transmembrane domain are from MPL S505N, and in certain embodiments,the extracellular domain and transmembrane domain of MPL S505N providedin Table 6 except that the recognition and elimination domain isoptional.

In another aspect, provided herein is an isolated polynucleotidecomprising one or more nucleic acid sequences, wherein:

-   -   a first nucleic acid sequence of the one or more nucleic acid        sequences encodes a chimeric polypeptide comprising in amino to        carboxy orientation,        -   a) a transmembrane domain from a type I transmembrane            protein; and        -   b) a first intracellular domain selected from an            intracellular domain of a gene having a first intracellular            domain of a selected polypeptide identified in Tables 11-16            wherein said chimeric polypeptide promotes cell            proliferation of B cells, T cells, and/or NK cells.    -   wherein said chimeric polypeptide promotes cell proliferation of        PBMCs, and in illustrative embodiments, of B cells, T cells,        and/or NK cells. As shown in the attached Example 18, such        chimeric polypeptides are capable of promoting cell        proliferation of PBMCs in the absence of exposure of the PBMCs        to IL-2 during culturing, for example in the absence of adding        IL-2 to culture media of PBMCs (e.g. B cells, T cells, or NK        cells) that express a nucleic acid encoding the chimeric        polypeptide.

In one embodiment of the aspect in the paragraph immediately above andany embodiment of this aspect herein below in this section, the firstintracellular domain is an intracellular domain of a gene having a firstintracellular domain in Tables 11-16, and in other illustrativeembodiments one of the first 50, 25, or 10 constructs listed in Tables11-16, and in further illustrative embodiments, one of the first 50, 25,or 10 constructs listed in two, three, four, or five of six tables of,or all of Tables 11-16. In one embodiment, the first intracellulardomain is an intracellular domain identified in Table 17. Table 17identifies constructs that promoted proliferation of PBMCs between day 7and day 21 or day 35 where a second intracellular domain was not presenton the construct. In some subembodiments of this embodiment, thechimeric polypeptide comprises a combination of transmembrane domain andintracellular domain, with or without an extracellular domain comprisinga dimerizing motif, of Table 17.

In one embodiment of the aspect in the paragraph immediately above andany embodiment of this aspect herein below in this section, the firstnucleic acid sequence further encodes an extracellular domain, and inillustrative embodiments, the extracellular domain comprises adimerizing motif. In any aspects or embodiments wherein theextracellular domain of a CLE comprises a dimerizing motif, thedimerizing motif can be selected from the group consisting of: a leucinezipper motif-containing polypeptide, CD69, CD71, CD72, CD96, Cd105,Cd161, Cd162, Cd249, CD271, and Cd324, as well as mutants and/or activefragments thereof that retain the ability to dimerize. In any of theaspects and embodiments herein wherein the extracellular domain of a CLEcomprises a dimerizing motif, the dimerizing motif can require adimerizing agent, and the dimerizing motif and associated dimerizingagent can be selected from the group consisting of: FKBP and rapamycinor analogs thereof, GyrB and coumermycin or analogs thereof, DHFR andmethotrexate or analogs thereof, or DmrB and AP20187 or analogs thereof,as well as mutants and/or active fragments of the recited dimerizingproteins that retain the ability to dimerize.

In illustrative embodiments of any aspects or embodiments herein whereinthe extracellular domain of a CLE comprises a dimerizing motif, theextracellular domain can comprises a leucine zipper motif. In someembodiments, the leucine zipper motif is from a jun polypeptide, forexample c-jun. In certain embodiments the c-jun polypeptide is the c-junpolypeptide region of ECD-11.

In one embodiment of this aspect, the first nucleic acid sequencefurther encodes a second intracellular domain, wherein the secondintracellular domain can be upstream (i.e. 5′) from the firstintracellular domain, or in illustrative embodiments the secondintracellular domain is downstream from the first intracellular domain.In a subembodiment of this embodiment, the first intracellular domainand the second intracellular domain combination comprises a firstintracellular domain of the selected polypeptide with any intracellulardomain of any gene identified in the second intracellular domains of anycandidate polypeptide in Tables 11-16, and in illustrative embodimentsin Tables 11-16, and in other illustrative embodiments one of the first50, 25, or 10 constructs listed in Tables 11-16, and in furtherillustrative embodiments, one of the first 50, 25, or 10 constructslisted in two, three, four, or five of tables of, or all of Tables11-16. In a subembodiment of this embodiment, the first intracellulardomain and the second intracellular domain combination comprise thefirst intracellular domain and the second intracellular domaincombination of the selected polypeptide.

In one embodiment of this aspect, the chimeric polypeptide comprises thetransmembrane domain, the first intracellular domain, and the secondintracellular domain of the selected polypeptide. In one embodiment ofthis aspect, the chimeric polypeptide comprises the extracellular andtransmembrane domain, the first intracellular domain, and the secondintracellular domain of the selected polypeptide, wherein theextracellular domain optionally comprises a recognition or eliminationdomain.

In a related subembodiment, the chimeric polypeptide comprises theextracellular and transmembrane domain, the first intracellular domain,and the second intracellular domain of the selected polypeptide exceptthat the extracellular and transmembrane domain optionally comprises arecognition or elimination domain, or does not comprise a recognition orelimination domain. In a related subembodiment, the chimeric polypeptidecomprises the extracellular and transmembrane domain, the firstintracellular domain, and the second intracellular domain of theselected polypeptide except that the extracellular and transmembranedomain comprises a recognition or elimination domain of the selectedpolypeptide, or another recognition or elimination domain.

In another embodiment of this aspect, the polynucleotide furthercomprises a second nucleic acid sequence that encodes a chimeric antigenreceptor (CAR) comprising an antigen-specific targeting region (ASTR), atransmembrane domain, and an intracellular activating domain. In asubembodiment of this aspect, the first intracellular domain and thesecond intracellular domain combination are the first intracellulardomain and the second intracellular domain combination of the selectedpolypeptide, and the selected polypeptide is identified in Tables 11-16,and in other illustrative embodiments one of the first 50, 25, or 10constructs listed in Tables 11-16, and in further illustrativeembodiments, one of the first 50, 25, or 10 constructs listed in two,three, four, or five of tables of, or all of Tables 11-16. In anothersubembodiment of this embodiment, the chimeric polypeptide comprises theextracellular and transmembrane domain, the first intracellular domain,and the second intracellular domain of the selected polypeptide, and theselected polypeptide is identified in Tables 11-16, or in illustrativeembodiments in one of the first 50, 25, or 10 constructs listed inTables 11-16, an in further illustrative embodiments, one of the first50, 25, or 10 constructs listed in two, three, four, or five of tablesof, or all of Tables 11-16. In a related subembodiment, the chimericpolypeptide comprises the extracellular and transmembrane domain, thefirst intracellular domain, and the second intracellular domain of theselected polypeptide except that the extracellular and transmembranedomain optionally comprises a recognition and elimination domain, andwherein the recognition or elimination domain is a recognition orelimination domain of the extracellular domain of the selectedpolypeptide, or another recognition or elimination domain, and whereinthe selected polypeptide is identified in Tables 11-16.

In another aspect, provided herein is an isolated polynucleotidecomprising one or more nucleic acid sequences, wherein:

-   -   a first nucleic acid sequence of the one or more nucleic acid        sequences encodes a chimeric polypeptide comprising in amino to        carboxy orientation,    -   a) a transmembrane domain from a type I transmembrane protein;        and    -   b) a first intracellular domain selected from an intracellular        domain of LEPR, MYD88, IFNAR2, MPL, IL18R1, IL13RA2, IL10RB,        IL23R, or CSF2RA, wherein said chimeric polypeptide promotes        cell proliferation of PBMCs, and in illustrative embodiments, of        B cells, T cells, and/or NK cells.

In one embodiment of the aspect in the paragraph immediately above andany embodiment of this aspect herein below in this section, the firstnucleic acid sequence further encodes an extracellular domain, and inillustrative embodiments, the extracellular domain comprises adimerizing motif. In illustrative embodiments of this aspect, theextracellular domain comprises a leucine zipper. In some embodiments,the leucine zipper is from a jun polypeptide, for example c-jun. Incertain embodiments the c-jun polypeptide is the c-jun polypeptideregion of ECD-11.

In another embodiment of this aspect where the first intracellulardomain is selected from an intracellular domain of LEPR, MYD88, IFNAR2,MPL, IL18R1, IL13RA2, IL10RB, IL23R, or CSF2RA, the first nucleic acidsequence further encodes a second intracellular domain, wherein thesecond intracellular domain can be upstream (i.e. 5′) from the firstintracellular domain, or in illustrative embodiments the secondintracellular domain is downstream from the first intracellular domain.In a subembodiment of this embodiment, the second intracellular domainis from a gene of a second intracellular domain in a selectedpolypeptide identified in any table of Example 18 as yielding anenrichment at day 21 versus day 7 above 0, 1, or 2, wherein the firstintracellular domain is from the gene of the first intracellular domainof the selected polypeptide. In a further subembodiment of thissubembodiment or embodiment, the first intracellular domain and thesecond intracellular domain combination are the first intracellulardomain and the second intracellular domain from a selected chimericpolypeptide identified in any table of Example 18 as yielding anenrichment at day 21 versus day 7 above 0, 1, or 2. In anothersubembodiment of the embodiment or subembodiments in this paragraph, thefirst intracellular domain is from a first gene of a selected firstintracellular domain of a selected polypeptide in Tables 11-16 and thesecond intracellular domain is from a second gene of a selected secondintracellular domain of the selected polypeptide. In anothersubembodiment of the embodiment or subembodiments in this paragraph, thefirst intracellular domain is from a selected polypeptide in Tables11-16 and the second intracellular domain is from the selectedpolypeptide. In another embodiment of this aspect or any embodimentherein, the polynucleotide further comprises a second nucleic acidsequence that encodes a chimeric antigen receptor (CAR) comprising anantigen-specific targeting region (ASTR), a transmembrane domain, and anintracellular activating domain.

In one embodiment of the aspect and one subembodiment or furthersubembodiment of the embodiments and subembodiments in the immediatelypreceding paragraph that comprise a second intracellular domain, thefirst intracellular domain is an intracellular domain of LEPR, MYD88,IFNAR2, and MPL. In another embodiment, the first intracellular domainis an intracellular domain from LEPR, IFNAR2, and MPL. In anotherembodiment, the first intracellular domain is other than MyD88. Inanother embodiment, the first intracellular domain is other than MyD88and the second intracellular domain is other than CD40. In asubembodiment of this the embodiments in this paragraph, thepolynucleotide further comprises a second nucleic acid sequence thatencodes a chimeric antigen receptor (CAR) comprising an antigen-specifictargeting region (ASTR), a transmembrane domain, and an intracellularactivating domain.

In one embodiment of this aspect of the invention, MPL is the firstintracellular domain and the second intracellular domain is from a geneof a second intracellular domain in a selected polypeptide identified inTables 11-16. In a further subembodiment of this embodiment, the firstintracellular domain and the second intracellular domain combinationcomprise a first intracellular domain and a second intracellular domainfrom a selected chimeric polypeptide identified in Tables 11-16. In somesubembodiments of the these embodiments, the orientation of the firstintracellular domain and the second intracellular domain is reversedsuch that the carboxy terminal residue of the second domain is bound tothe first domain or to a spacer between the second domain and the firstdomain.

In one embodiment of this aspect of the invention, LEPR is the firstintracellular domain and the second intracellular domain is from a geneof a second intracellular domain in a selected polypeptide identified inTables 11-16. In a further subembodiment of this embodiment, the firstintracellular domain and the second intracellular domain combinationcomprise a first intracellular domain and a second intracellular domainfrom a selected chimeric polypeptide identified in Tables 11-16. In somesubembodiments of the these embodiments, the orientation of the firstintracellular domain and the second intracellular domain is reversedsuch that the carboxy terminal residue of the second domain is bound tothe first domain or to a spacer between the second domain and the firstdomain.

In one embodiment of this aspect of the invention, IFNAR2 is the firstintracellular domain and the second intracellular domain is from a geneof a second intracellular domain in a selected polypeptide identified inTables 11-16. In a further subembodiment of this embodiment, the firstintracellular domain and the second intracellular domain combinationcomprise a first intracellular domain and a second intracellular domainfrom a selected chimeric polypeptide identified in Tables 11-16. In somesubembodiments of the these embodiments, the orientation of the firstintracellular domain and the second intracellular domain is reversedsuch that the carboxy terminal residue of the second domain is bound tothe first domain or to a spacer between the second domain and the firstdomain.

In one embodiment of this aspect of the invention, MyD88 is the firstintracellular domain and the second intracellular domain is from a geneof a second intracellular domain in a selected polypeptide identified inTables 11-16. In a further subembodiment of this embodiment, the firstintracellular domain and the second intracellular domain combinationcomprise a first intracellular domain and a second intracellular domainfrom a selected chimeric polypeptide identified in Tables 11-16. In somesubembodiments of the these embodiments, the orientation of the firstintracellular domain and the second intracellular domain is reversedsuch that the carboxy terminal residue of the second domain is bound tothe first domain or to a spacer between the second domain and the firstdomain.

In some embodiments of isolated polynucleotide aspects above wherein thefirst intracellular domain is selected from an intracellular domain ofLEPR, MYD88, IFNAR2, IL17RD, IL17RE, ILIRAP, IL23R, and MPL, thetransmembrane domain is selected from a transmembrane domain of CD40,CD8B, CRLF2, CSF2RA, GCGR2C, ICOS, IFNAR1, IFNGR1, IL10RB, IL18R1,IL18RAP, IL3RA, and PRLR. In some illustrative embodiments, thetransmembrane domain is a transmembrane domain of CD40 or ICOS. In somesubembodiments, the first intracellular domain is from LEPR, MYD88,IFNAR2, and MPL. For example, in some embodiments the transmembranedomain is a transmembrane domain from CD40 and the first intracellulardomain is an intracellular domain from MPL.

In some embodiments of isolated polynucleotide aspects above wherein thefirst intracellular domain is selected from an intracellular domain ofLEPR, MYD88, IFNAR2, IL17RD, IL17RE, ILIRAP, IL23R, and MPL, the secondintracellular domain is from CD40, CD79B, or CD27. In somesubembodiments, the first intracellular domain is from LEPR, MYD88,IFNAR2, and MPL. For example, in some embodiments, the firstintracellular domain is MPL and the second intracellular domain is CD40.In all embodiments in this paragraph, the order of the firstintracellular domain and the second intracellular domain on thepolypeptide can be reversed.

In another aspect, provided herein is an isolated polynucleotidecomprising one or more nucleic acid sequences, wherein:

-   -   a first nucleic acid sequence of the one or more nucleic acid        sequences encodes a chimeric polypeptide comprising in amino to        carboxy orientation,        -   a) a transmembrane domain from a type I transmembrane            protein; and        -   b) a first intracellular domain selected from an            intracellular domain of a gene having a first intracellular            domain of a selected polypeptide identified in Tables 11-16,            wherein said chimeric polypeptide promotes cell            proliferation of B cells, T cells, and/or NK cells.    -   wherein said chimeric polypeptide promotes cell proliferation of        PBMCs, and in illustrative embodiments, of B cells, T cells,        and/or NK cells. In some embodiments, the NK cells are NKT        cells.

In one embodiment of the aspect in the paragraph immediately above andany embodiment of this aspect herein below in this section, the firstnucleic acid sequence further encodes an extracellular domain, and inillustrative embodiments, the extracellular domain comprises adimerizing motif. In any aspects or embodiments wherein theextracellular domain of a CLE comprises a dimerizing motif, thedimerizing motif can be selected from the group consisting of: a leucinezipper motif-containing polypeptide, CD69, CD71, CD72, CD96, Cd105,Cd161, Cd162, Cd249, CD271, and Cd324, as well as mutants and/or activefragments thereof that retain the ability to dimerize. In any of theaspects and embodiments herein wherein the extracellular domain of a CLEcomprises a dimerizing motif, the dimerizing motif can require adimerizing agent, and the dimerizing motif and associated dimerizingagent can be selected from the group consisting of: FKBP and rapamycinor analogs thereof, GyrB and coumermycin or analogs thereof, DHFR andmethotrexate or analogs thereof, or DmrB and AP20187 or analogs thereof,as well as mutants and/or active fragments of the recited dimerizingproteins that retain the ability to dimerize.

In illustrative embodiments of any aspects or embodiments herein whereinthe extracellular domain of a CLE comprises a dimerizing motif, theextracellular domain can comprises a leucine zipper motif. In someembodiments, the leucine zipper motif is from a jun polypeptide, forexample c-jun. In certain embodiments the c-jun polypeptide is the c-junpolypeptide region of ECD-11.

In one embodiment of this aspect, the first nucleic acid sequencefurther encodes a second intracellular domain, wherein the secondintracellular domain can be upstream (i.e. 5′) from the firstintracellular domain, or in illustrative embodiments the secondintracellular domain is downstream from the first intracellular domain.In a subembodiment of this embodiment, the first intracellular domainand the second intracellular domain combination comprises a firstintracellular domain of the selected polypeptide with any intracellulardomain of any gene identified in the second intracellular domains of anycandidate polypeptide in Tables 11-16. In a subembodiment of thisembodiment, the first intracellular domain and the second intracellulardomain combination comprise the first intracellular domain and thesecond intracellular domain combination of the selected polypeptide.

In one embodiment of this aspect, the chimeric polypeptide comprises thetransmembrane domain, the first intracellular domain, and the secondintracellular domain of the selected polypeptide. In one embodiment ofthis aspect, the chimeric polypeptide comprises the extracellular andtransmembrane domain, the first intracellular domain, and the secondintracellular domain of the selected polypeptide, wherein theextracellular domain optionally comprises a recognition or eliminationdomain.

In a related subembodiment, the chimeric polypeptide comprises theextracellular and transmembrane domain, the first intracellular domain,and the second intracellular domain of the selected polypeptide exceptthat the extracellular and transmembrane domain optionally comprises arecognition or elimination domain, or does not comprise a recognition orelimination domain. In a related subembodiment, the chimeric polypeptidecomprises the extracellular and transmembrane domain, the firstintracellular domain, and the second intracellular domain of theselected polypeptide except that the extracellular and transmembranedomain comprises a recognition or elimination domain of the selectedpolypeptide, or another recognition or elimination domain.

In another embodiment of this aspect, the polynucleotide furthercomprises a second nucleic acid sequence that encodes a chimeric antigenreceptor (CAR) comprising an antigen-specific targeting region (ASTR), atransmembrane domain, and an intracellular activating domain. In asubembodiment of this aspect, the first intracellular domain and thesecond intracellular domain combination are the first intracellulardomain and the second intracellular domain combination of the selectedpolypeptide, and the selected polypeptide is identified in Tables 11-16.In another subembodiment of this embodiment, the chimeric polypeptidecomprises the extracellular and transmembrane domain, the firstintracellular domain, and the second intracellular domain of theselected polypeptide, and the selected polypeptide is identified inTables 11-16. In a related subembodiment, the chimeric polypeptidecomprises the extracellular and transmembrane domain, the firstintracellular domain, and the second intracellular domain of theselected polypeptide except that the extracellular and transmembranedomain optionally comprises a recognition and elimination domain, andwherein the recognition or elimination domain is a recognition orelimination domain of the extracellular domain of the selectedpolypeptide, or another recognition or elimination domain, and whereinthe selected polypeptide is identified in Tables 11-16.

In another aspect, provided herein is an isolated polynucleotidecomprising one or more nucleic acid sequences, wherein:

-   -   a first nucleic acid sequence of the one or more nucleic acid        sequences encodes a chimeric polypeptide comprising in amino to        carboxy orientation,        -   c) a transmembrane domain from a type I transmembrane            protein; and        -   d) a first intracellular domain selected from an            intracellular domain of LEPR, MYD88, IFNAR2, IL17RD, IL17RE,            ILIRAP, IL23R, and MPL, wherein said chimeric polypeptide            promotes cell proliferation of PBMCs, and in illustrative            embodiments, of B cells, T cells, and/or NK cells.

In one embodiment of the aspect in the paragraph immediately above andany embodiment of this aspect herein below in this section, the firstnucleic acid sequence further encodes an extracellular domain, and inillustrative embodiments, the extracellular domain comprises adimerizing motif. In illustrative embodiments of this aspect, theextracellular domain comprises a leucine zipper. In some embodiments,the leucine zipper is from a jun polypeptide, for example c-jun. Incertain embodiments the c-jun polypeptide is the c-jun polypeptideregion of ECD-11.

In another embodiment of this aspect where the first intracellulardomain is selected from an intracellular domain of LEPR, MYD88, IFNAR2,IL17RD, IL17RE, ILIRAP, IL23R, and MPL, the first nucleic acid sequencefurther encodes a second intracellular domain, wherein the secondintracellular domain can be upstream (i.e. 5′) from the firstintracellular domain, or in illustrative embodiments the secondintracellular domain is downstream from the first intracellular domain.In a subembodiment of this embodiment, the second intracellular domainis from a gene of a second intracellular domain in a selectedpolypeptide identified in any table of Example 18 as yielding anenrichment at day 21 versus day 7 above 0, 1, or 2, wherein the firstintracellular domain is from the gene of the first intracellular domainof the selected polypeptide. In a further subembodiment of thissubembodiment or embodiment, the first intracellular domain and thesecond intracellular domain combination are the first intracellulardomain and the second intracellular domain from a selected chimericpolypeptide identified in any table of Example 18 as yielding anenrichment at day 21 versus day 7 above 0, 1, or 2. In anothersubembodiment of the embodiment or subembodiments in this paragraph, thefirst intracellular domain is from a first gene of a selected firstintracellular domain of a selected polypeptide in Tables 11-16 and thesecond intracellular domain is from a second gene of a selected secondintracellular domain of the selected polypeptide. In anothersubembodiment of the embodiment or subembodiments in this paragraph, thefirst intracellular domain is from a selected polypeptide in Tables11-16 and the second intracellular domain is from the selectedpolypeptide. In another embodiment of this aspect or any embodimentherein, the polynucleotide further comprises a second nucleic acidsequence that encodes a chimeric antigen receptor (CAR) comprising anantigen-specific targeting region (ASTR), a transmembrane domain, and anintracellular activating domain.

In certain embodiments of any of the methods or isolated polynucleotideembodiments herein that include a chimeric polypeptide wherein saidchimeric polypeptide promotes cell proliferation of PBMCs, and inillustrative embodiments, of B cells, T cells, and/or NK cells, thefirst and/or second intracellular domain, in illustrative embodimentsthe first intracellular domain, is an intracellular domain from LEPR,MYD88, IFNAR2, MPL, IL18R1, IL13RA2, IL10RB, IL23R, or CSF2RA. In someembodiments, the first intracellular domain is from MPL. In exemplarysubembodiments, the chimeric polypeptide comprises an extracellulardomain comprising a dimerizing motif provided herein.

In certain embodiments of any of the methods or isolated polynucleotideembodiments herein that include a chimeric polypeptide wherein saidchimeric polypeptide promotes cell proliferation of PBMCs, and inillustrative embodiments, of B cells, T cells, and/or NK cells, thetransmembrane domain is from CD40, ICOS, FCGR2C, PRLR, IL3RA, or IL6ST.In some illustrative embodiments, the transmembrane domain is from CD40or ICOS. In exemplary subembodiments, the chimeric polypeptide comprisesan extracellular domain comprising a dimerizing motif provided herein.

In certain embodiments of any of the methods or isolated polynucleotideembodiments herein that include a chimeric polypeptide wherein saidchimeric polypeptide promotes cell proliferation of PBMCs, and inillustrative embodiments, of B cells, T cells, and/or NK cells, thefirst and/or second intracellular domain, in illustrative embodimentsthe second intracellular domain, is an intracellular domain from CD40,CD79B, TNFRSF4, TNFRSF9, TNFRSF14, FCGRA2, CD3G, or CD27. In someillustrative embodiments, the second intracellular domain is from CD40,CD79B, or CD27. In exemplary subembodiments, the chimeric polypeptidecomprises an extracellular domain comprising a dimerizing motif providedherein.

In certain embodiments of any of the methods or isolated polynucleotideembodiments herein that include a chimeric polypeptide wherein saidchimeric polypeptide promotes cell proliferation of PBMCs, and inillustrative embodiments, of B cells, T cells, and/or NK cells, thefirst and/or second intracellular domain, is an intracellular domain(either a P3 or P4 element) of any of the chimeric polypeptides inTables 11-16. In some embodiments the chimeric polypeptide comprises anyP2, P3, and P4 combination of any of the chimeric polypeptides in Tables11-16.

In an embodiment of any of the aspects and embodiments wherein thetransmembrane domain is a type I transmembrane protein, thetransmembrane domain can be a Type I cytokine receptor, a hormonereceptor, a T cell receptor, or a TNF-family receptor. In an embodimentof any of the aspects and embodiments wherein the chimeric polypeptidecomprises an extracellular domain and wherein the extracellular domaincomprises a dimerizing motif, the transmembrane domain can a Type Icytokine receptor, a hormone receptor, a T cell receptor, or aTNF-family receptor. In illustrative subembodiments of any of theseembodiments in this paragraph, the first intracellular domain isselected from an intracellular domain of LEPR, MYD88, IFNAR2, IL17RD,IL17RE, ILIRAP, IL23R, and MPL.

In an embodiment of any of the aspects and embodiments directed topolynucleotides comprising a nucleic acid sequence encoding a chimericpolypeptide that comprises an extracellular domain, the extracellulardomain can comprise a 1, 2, 3, or 4 amino acid spacer within 20 aminoacids of the carboxy terminus of the extracellular domain, and inillustrative embodiments, at the carboxy-terminus of the extracellulardomain such that the spacer separates the extracellular domain and thetransmembrane domain. In some embodiments, the 1, 2, 3, or 4 amino acidspacer comprises an alanine residue, and in some cases, only alanineresidues. In illustrative subembodiments for embodiments that comprisesaid spacer, the extracellular domain comprises a dimerizing motif, forexample a leucine zipper motif.

It will be understood that the first intracellular domain and the secondintracellular domain for any aspect and embodiment provided herein thatcomprises a chimeric polypeptide that promotes cell proliferation ofPBMCs, B cells, T cells, and/or NK cells, can include mutants and/orfragments of the recited genes provided that the chimeric polypeptideretains its ability to promote cell proliferation of PBMCs, B cells, Tcells, and/or NK cells. These mutants and/or fragments typically retainsignaling activity such that the chimeric polypeptide retains itssurvival and proliferation promoting activity. It will be understoodthat the first intracellular domain and the second intracellular domaincan be reversed in order for any aspect and embodiment provided hereinthat comprises a chimeric polypeptide that promotes cell proliferationof PBMCs, B cells, T cells, and/or NK cells. Thus, it will be understoodin these embodiment that the first intracellular domain is the secondintracellular domain and vice versa.

In any of the aspects and embodiments provided herein that comprisepolynucleotides that include a nucleic acid sequence encoding a chimericpolypeptide that promotes cell proliferation of PBMCs, B cells, T cells,and/or NK cells, the nucleic acid sequence can further encode arecognition or elimination domain. In some embodiments, the recognitionor elimination domain is a recognition domain of a monoclonal antibodyapproved biologic. In some embodiments, the recognition or eliminationdomain comprises a polypeptide that is recognized by an antibody thatrecognizes EGFR, or an epitope thereof. In some embodiments, therecognition or elimination domain comprises a polypeptide that isrecognized by an antibody that recognizes MYC, or an epitope thereof.

In any of the aspects and embodiments provided herein that comprisespolynucleotides that include a nucleic acid sequence encoding a chimericpolypeptide that promotes cell proliferation of PBMCs, B cells, T cells,and/or NK cells, the first nucleic acid sequence, and/or the secondnucleic acid sequence can be operatively linked to a first promoteractive in B cells, T cells and/or NK cells. In some illustrativeembodiments, the first promoter is active in T cell and/or NK cells.

In an embodiment of any of the isolated polynucleotide aspects andembodiments provided herein having a nucleic acid sequence that encode achimeric polypeptide that promotes cell proliferation of PBMCs, B cells,T cells, and/or NK cells, a linker can be present between thetransmembrane domain and the first intracellular domain and/or betweenthe first intracellular domain and the second intracellular domain, forpolynucleotide embodiments that comprise a second intracellular domain.In some embodiments, a linker of between 1 and 4 alanines is presentbetween the extracellular domain and the transmembrane domain forembodiments comprising an extracellular domain.

In an embodiment of any of the isolated polynucleotide or vector aspectsand embodiments provided herein that comprises a first nucleic acidsequence that encodes a chimeric polypeptide that promotes cellproliferation of PBMCs, B cells, T cells, and/or NK cells, and a secondnucleic acid sequence that encodes a chimeric antigen receptor (CAR)comprising an antigen-specific targeting region (ASTR), a transmembranedomain, and an intracellular activating domain, the first nucleic acidsequence and the second nucleic acid sequence can be separated by aribosomal skip sequence. In some embodiments, the ribosomal skipsequence is F2A, E2A, P2A, or T2A.

In an embodiment of any of the isolated polynucleotide aspects andembodiments provided herein that encode a chimeric polypeptide thatpromotes cell proliferation of PBMCs, B cells, T cells, and/or NK cells,said polynucleotide further comprises one or more of a Kozak-typesequence, a WPRE element, and a double stop codon or a triple stopcodon, wherein one or more of the stop codons of the double stop codonor the triple stop codon define a termination of a reading from of atleast one of the one or more transcriptional units. In certainembodiments, the polynucleotide further comprises a Kozak-type sequenceselected from CCACCAUG(G), CCGCCAT/UG(G), GCCGCCGCCAT/UG(G), orGCCGCCGCCAT/UG. In certain embodiments both the −3 and +4 nucleotidesrelative to a start codon of the first nucleic acid sequence comprise aG. In another embodiment, which can be combined with the precedingembodiment that comprises a Kozak-type sequence and/or the followingembodiment that includes triple stop codon, the polynucleotide comprisesa WPRE element. In some embodiments, the WPRE element is located 3′ of astop codon of the one or more transcriptional units and 5′ to a 3′ LTRof the polynucleotide. In another embodiment, which can be combined witheither or both of the preceding embodiments (i.e. an embodiment thepolynucleotide comprises a Kozak-type sequence and/or an embodimentwherein the polynucleotide comprises a WPRE), the one or moretranscriptional units terminates with one or more stop codons of adouble stop codon or a triple stop codon.

In one aspect, provided herein is a nucleic acid vector comprising anisolated polynucleotide of any of the isolated polynucleotide aspectsand embodiments provided herein that encode a chimeric polypeptide thatpromotes cell proliferation of PBMCs, B cells, T cells, and/or NK cells,

-   -   a. an origin of replication;    -   b. an antibiotic resistance gene; and/or    -   c. one or more viral and/or retroviral sequences selected from a        retroviral long terminal repeat (LTR), or active fragment        thereof, a retroviral cis-acting RNA packaging element, and/or a        retroviral central polypurine tract/central termination sequence        (CTS) element.

In certain embodiments of this aspect, the vector is a plasmid. In otherembodiments, the vector is a replication incompetent viral vector, forexample, a replication incompetent retroviral vector or particle. Inthese embodiments, the replication incompetent retroviral vector orparticle, can comprise a 5′ retroviral LTR, or active fragment thereof,a retroviral cis-acting RNA packaging element, and a 3′ retroviral LTR,or active fragment thereof, wherein the 5′ retroviral LTR and the 3′retroviral LTR flank the isolated polynucleotide. In some embodiments,said isolated polynucleotide is in reverse orientation to the nucleicacid sequence encoding the retroviral cis-acting RNA packaging element,the 5′ long terminal repeat, and/or the 3′ long terminal repeat.

In some aspects, provided herein is a replication incompetentrecombinant retroviral particle comprising a retroviral genomecomprising any of the aspects and embodiments herein that comprise anisolated polynucleotide that comprises a first nucleic acid sequencethat encodes a chimeric polypeptide that promotes cell proliferation ofPBMCs, B cells, T cells, and/or NK cells, and further comprising acapsid and an envelope. The retroviral particle can additionallycomprise any of the retroviral elements set out in this disclosure. Inone embodiment, the retrovirus genome further comprises a second nucleicacid sequence that encodes a chimeric antigen receptor (CAR) comprisingan antigen-specific targeting region (ASTR), a transmembrane domain, andan intracellular activating domain. In some embodiments, the firstnucleic acid sequence and the second nucleic acid sequence can beseparated by a ribosomal skip sequence. In some embodiments, theribosomal skip sequence is F2A, E2A, P2A, or T2A. In these embodiments,the first nucleic acid sequence and the second nucleic acid sequence aretypically on the same transcriptional unit.

In another aspect, provided herein is a mammalian cell wherein saidmammalian cell, for example human cell, is a B cell, T cell or NK cellcomprising an isolated polynucleotide, which optionally can beintegrated into the genome of the cell, according to any of the aspectsand embodiments herein that comprise an isolated polynucleotide thatcomprises a first nucleic acid sequence that encodes a chimericpolypeptide that promotes cell proliferation of PBMCs, B cells, T cells,and/or NK cells. In some embodiments, the isolated polynucleotide in themammalian cell further comprises a second nucleic acid sequence thatencodes a chimeric antigen receptor (CAR) comprising an antigen-specifictargeting region (ASTR), a transmembrane domain, and an intracellularactivating domain. In some embodiments, the first nucleic acid sequenceand the second nucleic acid sequence can be separated by a ribosomalskip sequence. In some embodiments, the ribosomal skip sequence is F2A,E2A, P2A, or T2A. In these embodiments, the first nucleic acid sequenceand the second nucleic acid sequence are typically on the sametranscriptional unit. In some embodiments, the cell is a T cell or NKcell, and in certain illustrative embodiments, the cell is a human Tcell.

In another aspect, provided herein is a packaging cell comprising anisolated polynucleotide according to any of the aspects and embodimentsherein that comprise an isolated polynucleotide, which optionally can beintegrated into the genome of the cell, that comprises a first nucleicacid sequence that encodes a chimeric polypeptide that promotes cellproliferation of PBMCs, B cells, T cells, and/or NK cells. In someembodiments, the isolated polynucleotide in the cell further comprises asecond nucleic acid sequence that encodes a chimeric antigen receptor(CAR) comprising an antigen-specific targeting region (ASTR), atransmembrane domain, and an intracellular activating domain. In someembodiments, the first nucleic acid sequence and the second nucleic acidsequence can be separated by a ribosomal skip sequence. In someembodiments, the ribosomal skip sequence is F2A, E2A, P2A, or T2A. Inthese embodiments, the first nucleic acid sequence and the secondnucleic acid sequence are typically on the same transcriptional unit. Insome embodiments, the cell is cell provided herein for generation ofretroviral particles.

In one aspect, provided herein is a method of promoting cellproliferation of a PBMC, for example a B cell, T cell, and/or NK cell,comprising transducing and/or genetically modifying the cell with avector that encodes a lymphoproliferative element, and in illustrativeembodiments a chimeric polypeptide according to any of the embodimentsprovided herein, wherein the chimeric polypeptide promotes PBMCproliferation. The method can include culturing the cell for at least 7,14, 21, 28, 35, 42, or 60 days. Typically, this culturing is performedin the absence of IL-2, for example after transduction with media thatcan include IL-2 in some embodiments.

In one aspect, provided herein is a method for transfecting, transducingand/or genetically modifying a B cell, T cell, and/or NK cell, which insome non-limiting embodiments, can be an allogeneic cell, an autologouscell, or other than an allogeneic cell, comprising contacting the cellwith an isolated polynucleotide according to any of the aspects andembodiments herein that comprise an isolated polynucleotide thatcomprises a first nucleic acid sequence that encodes a chimericpolypeptide that promotes cell proliferation of PBMCs, B cells, T cells,and/or NK cells (CLE aspects and embodiments). In some embodiments ofthis method aspect, the cell is a T cell and the isolated polynucleotidefurther comprises a second nucleic acid sequence that encodes a chimericantigen receptor (CAR) comprising an antigen-specific targeting region(ASTR), a transmembrane domain, and an intracellular activating domain.In other words, the method for transfecting, transducing and/orgenetically modifying a B cell, T cell, and/or NK cell can includecontacting the cell with isolated polynucleotides according to any ofthe CLE or CLE CAR embodiments herein. In some embodiments, the firstnucleic acid sequence and the second nucleic acid sequence can beseparated by a ribosomal skip sequence. In some embodiments, theribosomal skip sequence is F2A, E2A, P2A, or T2A. In these embodiments,the first nucleic acid sequence and the second nucleic acid sequence aretypically on the same transcriptional unit. In some embodiments, thecell is a T cell or NK cell, and in certain illustrative embodiments,the cell is a human T cell. The contacting is performed under effectiveconditions for transfecting, transducing and/or genetically modifyingthe cell. In some embodiments, the isolated polynucleotide at the timeof contacting is within a vector according to any of the vector aspectand embodiments provided herein. In some embodiments, the isolatedpolynucleotide is in a non-replicative retroviral particle at the timeof contacting.

In some embodiments of any of the aspects herein, the NK cells are NKTcells.

The following non-limiting examples are provided purely by way ofillustration of exemplary embodiments, and in no way limit the scope andspirit of the present disclosure. Furthermore, it is to be understoodthat any inventions disclosed or claimed herein encompass allvariations, combinations, and permutations of any one or more featuresdescribed herein. Any one or more features may be explicitly excludedfrom the claims even if the specific exclusion is not set forthexplicitly herein. It should also be understood that disclosure of areagent for use in a method is intended to be synonymous with (andprovide support for) that method involving the use of that reagent,according either to the specific methods disclosed herein, or othermethods known in the art unless one of ordinary skill in the art wouldunderstand otherwise. In addition, where the specification and/or claimsdisclose a method, any one or more of the reagents disclosed herein maybe used in the method, unless one of ordinary skill in the art wouldunderstand otherwise.

Examples Example 1. Generation of Riboswitches that Respond Specificallyto Nucleoside Analogue Antiviral Drugs

This example provides a method to screen libraries based on naturalstructural riboswitches that bind guanosine and deoxyguanosine. Theseriboswitches were used as scaffolds to develop biased libraries for theselection of aptamers that bind specifically to a ligand nucleosideanalogue. Previously, isothermal titration calorimetry has been used toshow these natural riboswitches bind to their native ligands. Additionaltests showed a deoxyguanosine switch also interacted weakly with thenucleoside analogues acyclovir and penciclovir, leading to the re-designof this sequence into a new library. The single-stranded regions of theriboswitch were targeted for mutation and variant sequences thatspecifically respond to acyclovir or penciclovir were selected for.

Materials

Selection components guanine, guanosine, deoxyguanosine, acyclovir, andpenciclovir were ordered from Sigma-Aldrich (St. Louis, MO). Acyclovirwas the initial target while penciclovir was a special interest analyteused in latter rounds and guanine, guanosine, and deoxyguanosine wereused as counter-targets. Graphene oxide (GrO), to be used as thepartitioning medium, was purchased from Angstron Materials (Dayton, OH).HEPES (pH 7.3) and MgCl₂ were purchased from Amersco LLC. (Solon, OH).KCl was purchased from Teknova (Hollister, CA). Selection buffer wasprepared at 5× (1× as 50 mM HEPES, 100 mM KCl, 20 mM MgCl₂, pH 7.3).Targets, counter-targets, and oligos were reconstituted in nuclease-freewater for preliminary analysis and aptamer screening. Aliquots wereprepared for all targets and stored at −20° C. to maximize shelf life.

Generation of the Aptamer Library

The initial aptamer library template was synthesized by IBA GmbH(Gottingen, Germany) as the reverse complement of the sequences in FIG.14 . In FIG. 14 , the nucleotides in boxes are single-stranded in theknown sequences, with “mutations” introduced during synthesis to allowfor better binding to analogues of the original targets. For nucleotideswithin the boxes outlined with solid lines, substitution mutations wereallowed; for nucleotides within the boxes outlined with dashed lines,substitution mutations as well as insertions or deletions were allowed.Primers were synthesized by IDT (Coralville, IA) as single-stranded DNA.T7 primer (SEQ ID NO:240) was combined with library template sequencesfor primer extension with Titanium Taq DNA polymerase (Clontech;Mountain View, CA). Primer-extended material was transcribed using theAmpliscribe T7 High Yield Transcription Kit (Epicentre; Madison, WI) andthen purified on 10% denaturing polyacrylamide gel electrophoresis(PAGE) with 8 M urea before use in selection. During selection, thelibrary was reverse-transcribed using SuperScript IV ReverseTranscriptase (Invitrogen; Carlsbad, CA) using reverse primer (SEQ IDNO:241) and amplified using Titanium Taq DNA polymerase (Clontech;Mountain View, CA). The aptamer with SEQ ID NO:248 had a J2-3 loopvariation of −3 to −1 and a diversity of ˜2.25×10m. The aptamer with SEQID NO:250 had a J2-3 loop variation of 0 (native) to +5 and a diversityof ˜9.38×10¹⁴. The two oligonucleotides (SEQ ID NOs:249 and 250) weremixed at a ratio of 1:4160 to produce equimolar diversity in thecombined library pool, with a total diversity of ˜9.38×10¹³.

Library Screening

Library screening was conducted using a graphene oxide-SystematicEvolution of Ligands by EXponential enrichment (GO-SELEX) approach (FIG.15 ) (Park et al., 2012), taking advantage of the 7L-7L interaction thatgrants graphene oxide a high affinity for single-stranded nucleic acids(Zeng et al., 2015). The goal was to select sequences that did notinteract with the 1× selection buffer or with the counter-targets(guanine, guanosine, and deoxyguanosine) but did bind to the positivetarget acyclovir.

For each round, a given amount of library was first refolded in 1×selection buffer (5-minute denaturing at 90° C., 5 minutes at 4° C.,then room temperature). The counter-targets were then added to refoldedlibraries and incubated for 30 minutes at 37° C. The exceptions to thiswere rounds 1 and 2, where the counter-targets were only briefly (<1minute) included to help load the library onto the GrO. After allowingthe library to interact with the counter-targets and buffer components,unbound library was loaded onto GrO (mass equal to 100 times the mass ofthe library at the start of the round) over the course of a 10-minuteincubation at 37° C. The solution was then centrifuged at 7,000×g tosediment the GrO. The supernatant, which contained sequences bound tothe counter-targets and/or to the buffer, was removed. The sediment wasthen washed twice with 200 L 1× selection buffer, centrifuging at7,000×g and removing the supernatant after each wash. A positivetarget-containing solution was then added and allowed to elute libraryfrom the GrO under the conditions indicated in Table 1 for up to 60minutes at 37° C., essentially allowing the target to compete withgraphene oxide for library binding. Sequences that bound more stronglyto the target would desorb from graphene oxide and remain bound to thetarget at the end of the incubation. A final centrifugation stepseparated the released material, located in the supernatant, from thenon-responsive library that remained bound to the graphene oxide.

After positive selection, the recovered RNA purified using 10%denaturing PAGE with 8 M Urea, was then quantified using aspectrophotometer reading (Table 1), reverse-transcribed withSuperScript IV, and amplified using PCR with Titanium Taq DNApolymerase. Amplification products were transcribed into RNA for thenext round of selection.

Three tiers of stringency were implemented over the course of selection(Table 1). The first two rounds of selection did not include screeningagainst counter-targets to maximize library loading onto GrO.Additionally, a large excess of acyclovir was used in positiveincubations to maximize library recovery, thus the low-stringencydesignation. Counter-target incubations were introduced after libraryrecovery was achieved, as middle-stringency conditions. The ratio ofacyclovir to library was also reduced during these three rounds toincrease library competition for binding to target. Once greater than10% recovery was achieved, the final rounds of high-stringency selectionwere implemented. Counter-targets/library ratio remained high andpositive target/library ratio was brought to 1:1 while positiveincubation time was reduced, to select for faster binding sequences.Once library recovery was shown to remain over 10% after more than tworounds of the high-stringency conditions, parallel assessments wereconducted.

TABLE 1 Selection and Assessment Conditions. Conditions used for eachround of selection or incubation, with recovery as the ratio betweenrecovered sample and input library for each round. Library enrichmentwas monitored over the course of selection. Generation Library:X-Targets(+) Incubation (Stringency) (30-min inc.) Library:(+) Target Time (min)Recovery (%) G0/R1 (low) 1:1000* 1:1000 60 0.43 G1/R2 (low) 1:1000*1:1000 60 2.00 G2/R3 (middle) 1:1000 1:500 60 3.60 G3/R4 (middle) 1:10001:100 60 8.73 G4/R5 (middle) 1:1000 1:10 60 10.20 G5/R6 (high) 1:10001:1 60 12.00 G6/R7 (high) 1:1000 1:1 60 8.60 G7/R8 (high) 1:1000 1:1 609.72 G8/R9 (high) 1:1000 1:1 30 20.08 G9/R10 (high) 1:1000 1:1 30 10.62G10(−)^(†) (parallel 1) — — 30 3.74 G10(X)^(†) (parallel 1) 1:40 — 303.60 G10(+)^(†) (parallel 1) — 1:4 30 14.14 G10(P)^(†) (parallel 1) —1:4 30 5.46 G11(−)^(‡) (parallel 2) — — 30 4.60 G11(X)^(‡) (parallel 2)1:40 — 30 5.26 G11(+)^(†) (parallel 2) — 1:2 30 9.34 G11(P)^(‡)(parallel 2) — 1:4 30 6.32 *Counter-targets used for loading, notextended incubation. ^(†)Pre-loading incubation conducted with pooledcounter-targets. ^(‡)Pre-loading incubation conducted with positivetarget acyclovir. This was done to minimize the recovery ofcross-reactive species. The following abbreviations are used in thistable: “X-Targets” are counter-targets; “(+) Target” is acyclovir orpenciclovir; “(+) Incubation Time (min)” is the time the “Library:(+)Target” solution was incubated on the GrO. G0 is Generation 0 and so on;R1 is Round 1 and so on. For the parallel assessment (parallel 1 andparallel 2) the incubations were performed with: (−) 1X selection bufferonly, (X) counter-targets in 1X selection buffer, (+) acyclovir in 1Xselection buffer, and (P) penciclovir in 1X selection buffer.

For the two parallel assessments, library to be assessed was dividedinto four equal amounts for preparation and refolding as above (FIG. 16). For each condition, 50 pmoles of library were combined with 1×selection buffer, refolded (90° C. for 5 minutes, 4° C. for 5 minutes),and then incubated with 200 L of 10 μM combined counter-targets in 1×selection buffer for 30 minutes at 37° C. These samples were then loadedonto an amount of graphene oxide equal to 100 times the mass of libraryin the sample and incubated for 10 minutes at 37° C. and then washedtwice with 200 μL of 1× selection buffer as before. The loaded grapheneoxide samples were then incubated in parallel with 200 μL of theappropriate assessment condition (1× selection buffer only, 10 μM pooledcounter-targets, 1 μM penciclovir, 1 μM acyclovir for the first parallelassessment, or 0.5 μM acyclovir for the second parallel assessment; inTable 1 these conditions are shown as: (−); (X); (P); (+); and (+),respectively) in 1× selection buffer for 30 minutes at 37° C. A finalcentrifugation step separated desorbed responsive library fromnon-responsive graphene oxide-bound library. The responsive librarieswere quantified using spectrophotometric reading (Table 1), verifiedusing 10% denaturing PAGE with 8 M urea, and prepared for a secondparallel assessment. This follow-up assessment continued to usecounter-targets for the positive sample's pre-loading incubation, bututilized positive target acyclovir for each other samples'pre-incubation. This was done to minimize representation ofcross-reactive sequences in a given sample (i.e. responsive tocounter-targets in the positive sample, responsive to acyclovir in thenegative, counter-targets, or penciclovir samples). Material recoveredfrom the second parallel assessment was quantified usingspectrophotometric reading (Table 1), verified using 10% denaturing PAGEwith 8 M urea, and prepared for sequencing by reverse transcription andPCR to generate double-stranded DNA.

Sequencing

The initial library was subjected to over 10 rounds of GrO-basedselection and parallel assessment (Table 1). The GO-SELEX process isdesigned to enrich for sequences over multiple rounds of selection thatbind to the given targets of interest and remove sequences that bind tothe non-target compounds or buffer components. As a result, thepopulations to be sequenced are expected to contain multiple copies ofpotential aptamer candidates.

The Illumina MiSeq system (San Diego, CA) was implemented to sequencethe aptamer libraries after parallel assessment using a single-end readtechnique. Deep sequencing and subsequent data analysis reduces thelarge number of screening rounds traditional SELEX requires, which mayintroduce error and bias due to the screening process (Schutze et al.,2011). Five samples were sequenced: the final generation library thatresponded to acyclovir, the final generation library that responded tothe counter-targets, the final generation library that responded to 1×selection buffer (negative condition), the penultimate generationlibrary that responded to acyclovir, and the final generation librarythat responded to the additional target of interest, penciclovir. Fromthese sets of data, sequence families were constructed at 95% homology(sequence similarity considering mutations, deletions, and insertion)for aptamer candidate identification. There were 1,711,535 raw sequences(124,600 unique sequences) from the library that responded to acyclovirand 2,074,832 raw sequences (110,149 unique sequences) from the librarythat responded to penciclovir.

Aptamer Candidate Selection

Sequence family construction focused primarily on sequence similarity.This means that a sequence's frequency in the positive target populationwas factored in, but greater emphasis was placed on the degree ofvariation between similar sequences, with 95% homology being the minimumrequirement (100% match over the entire sequence is not necessary tojoin a family, up to 2 bases can be mismatched, inserted, or deleted).One would therefore expect families with the greatest number of membersto rank highly as aptamer candidates. After families are constructed,consideration can be given to the relative presence of a family in agiven population—families that occur frequently in the negative andcounter-target populations are considered weaker candidates, as theydemonstrate a degree on non-specific interaction in binding to buffer orcounter-target components. Additionally, families that demonstrate ahigh rate of enrichment (i.e. large ratio between the final positivepopulation and penultimate positive population) improve their candidacy,as enrichment rate has been linked to the binding affinity of acandidate relative to the rest of the population (Levay et al., 2015;Wang et al., 2014). Under these conditions, several candidate familiesappeared to be strong candidates for binding acyclovir (Table 2) andpenciclovir.

TABLE 2 Candidate Family- SEQ Sequence ID % Identity Number NO: SequenceLength Consensus Wildtype 582-1 108 ACAGCTTAGCGTAATGGCTACTGACG 49 10080.77 CCGTCCAAACCTATTTACAGACT 582-2 109 ACAGCTTAGGATAATGGCTACTGACG 49 95.92 80.77 CCGTCCAAACCTATTTACAGACT 582-3 110ACAGCTTAGCATAATGGCTACTGACG 49  95.92 80.77 CCGTCCAAACCTATTCACAGACT 582-4111 ACAGCTTAGCATAATGGCTACTGACG 49  95.92 80.77 CCGTCCAAACCTATTGACAGACT582-5 112 ACAGCATAGCATAATGGCTACTGAC 49  95.92 82.69GCCGTCCAAACCTATTTACAGACT 582-6 113 ACAGCTTAGCATAATGGCTACTGACG 49  95.9280.77 CCGTCCAAACCTATGTACAGACT 582-7 114 ACAGCTAGCGTAATGGCTACTGACGC 48 97.96 80.77 CGTCCAAACCTATTTACAGACT 582-8 115 ACAGCTTAGCATTATGGCTACTGACG49  95.92 80.77 CCGTCCAAACCTATTTACAGACT 582-9 116ACAGTTAGCATAATGGCTACTGACGC 48  95.92 82.69 CGTCCAAACCTATTTACAGACT 582-10117 ACAGCTTAGCATAATGGCTACTGACG 49  95.92 80.77 CGGTCCAAACCTATTTACAGACT582-11 118 ACAGCTTAGCTTAATGGCTACTGACG 49  97.96 80.77CCGTCCAAACCTATTTACAGACT 582-12 119 ACAGCTTAGCATAATGGCTACTGACG 49  95.9280.77 CCGTCCAAACCCATTTACAGACT 582-13 120 ACAGCTTAGCATAATGGCTACTGACG 49 95.92 80.77 CCGTCCAAACCAATTTACAGACT 582-14 121ACAGCTTAGCATAATGGATACTGACG 49  95.92 80.77 CCGTCCAAACCTATTTACAGACT582-15 122 ACAGCTTAGCATTGTGGCTACTGACG 49  93.88 78.85CCGTCCAAACCTATTTACAGACT 582-16 123 ACAGGTTAGCATAATGGCTACCGAC 49  93.8882.69 GCCGTCCAAACCTATTTACAGACT 582-17 124 ACAGCTTAGCGTAATGGCTACTGACG 49 97.96 82.69 CCGCCCAAACCTATTTACAGACT 582-18 125ACAGCTTAGCATAATGGCTACTGACG 49  93.88 80.77 CCGTCCAAAACTATTTCCAGACT582-19 126 ACAGCCTAGCATAAGGGCTACTGAC 49  93.88 82.69GCCGTCCAAACCTATTTACAGACT 582-20 127 ACAGCTTAGCATAATGGCTACTGAGG 49  95.9280.77 CCGTCCAAACCTATTTACAGACT 582-21 128 ACAGCTTACCTTAATGGCTACTGACG 49 95.92 78.85 CCGTCCAAACCTATTTACAGACT 582-22 129ACAGCTTAGCATAATGGCTACCGACG 49  93.88 78.85 CTGTCCAAACCTATTTACAGACT582-23 130 ACAGCTTAGCGTAATGGCTACTGGCG 49  97.96 78.85CCGTCCAAACCTATTTACAGACT 582-24 131 ACAGCTTAGCATACTGGCTACTGACG 49  93.8882.69 CCGCCCAAACCTATTTACAGACT 582-25 132 ACAGCTTAGCATAATGGCTACTGACG 49 95.92 80.77 CCGTCCTAACCTATTTACAGACT 582-26 133ACAGGTTAGCATAATGCCTACTGACG 49  93.88 82.69 CCGTCCAAACCTATTTACAGACT582-27 134 ACAGCTTAGCATAATTGCTACTGACG 49  93.88 82.69CCGTTCAAACCTATTTACAGACT 582-28 135 ACAGCTTAGCATAAAGGCTACTGAC 49  95.9280.77 GCCGTCCAAACCTATTTACAGACT 582-29 136 ACAGCTTAGCGTAATGGCTACTGACG 49 95.92 80.77 CCGTCTAAACCTATTTCCAGACT 582-30 137ACAGGTTAGCATAATGGCTACTGACG 49  93.88 86.54 CCGTCCAAACCTATTTAGAGACT582-31 138 ACAGGGTAGCGTAATGGCTACTGAC 49  95.92 84.62GCCGTCCAAACCTATTTACAGACT 582-32 139 ACAGCGTAGCATAATGGCTACTGAC 49  93.8886.54 GCCGTTCAAACCTATTTACAGACT 582-33 140 ACAGCTTAGCATAATGGCTACTGACG 49 93.88 78.85 CCGTCCAAACTCATTTACAGACT 582-34 141ACAGCGTAGCATAGTGGCTACTGAC 49  93.88 82.69 GCCGTCCAAACCTATTTACAGACT582-35 142 ACAGCTTAGTGTAATGGCTACTGACG 49  95.92 76.92CTGTCCAAACCTATTTACAGACT 582-36 143 ACAGCTTAGCATAATGGCTACTGACG 49  93.8882.69 GCGTTCAAACCTATTTACAGACT 582-37 144 ACAGGTTAGCATAATGGCTACTGACG 49 93.88 84.62 CCGTCCAAACCTATTTATAGACT 582-38 145ACAGCTTAGCATAATGGCTACTGACG 48  91.84 80.77 CCGTCCAAACCTATTGTCGACT 582-39146 ACAGCTTAGCATAATGGCTACTGACG 48  95.92 80.77 CCGTCCAAACCTATTTACGACT582 222 ACAGNNTASBDTWVDKSMTACYGRS 49 — — ConsensusGSBGYYYWAAMYHATKBHBNGACT SequenceWhere the N at position 5 can be C, G, orno nucleotide, the N at position 6 can be A,C, G, T, or no nucleotide, and the N atposition 45 can be A or no nucleotide. 769-1 147ACAGGTCAGCATAATGTGCTAGTGCG 48 100 82.69 CCTTCAAACCTATTTAGAGACT 769-2 148ACAGGTCAGCATAATGTGCTAGTGCG 48  97.92 80.77 CCCTCAAACCTATTTAGAGACT 769-3149 ACAGGTTAGCATAATGTGCTATTGCG 48  95.83 84.62 CCTTCAAACCTATTTAGAGACT769-4 150 ACAGGTCAGCATAATGTGCTAGTGCG 48  97.92 80.77CATTCAAACCTATTTAGAGACT 769-5 151 ACAGGTTAGCATAATGTGCTAGTGCG 48  95.8384.62 CCTTCAAACCTATTTTGAGACT 769-6 152 ACAGGTTATCATAATGTGCTAGTGCG 48 95.83 82.69 CCTTCAAACCTATTTAGAGACT 769-7 153 ACAGGTTAGCATGATGTGCTAGTGCG48  95.83 82.69 CCTTCAAACCTATTTAGAGACT 769-8 154ACAGGTTAGCATAATGGGCTAGTGC 48  95.83 86.54 GCCTTCAAACCTATTTAGAGACT 769-9155 ACAGGTCAGCAAAATGTGCAAGTGC 48  95.83 78.85 GCCTTCAAACCTATTTAGAGACT769-10 156 ACAGGTCAGCATAATGTGCTAGTGCG 48  95.83 82.69CCTTCAAACCTATCTGGAGACT 769-11 157 ACAGCTTAGCATAATGTGCTAGTGCG 48  95.8382.69 CCTTCAAACCTATTTAGAGACT 769-12 158 ACAGGTCAGCATAATGTGCTAGTGCG 48 97.92 80.77 CCTTCAAACCTATTTACAGACT 769-13 159ACAGGTCAGCATAATGTGCTAGTGCG 48  97.92 80.77 CCTTCAAACATATTTAGAGACT 769-14160 ACAGGGTAGCATAATGTGCTAGTGC 48  95.83 86.54 GCCTTCAAACCTATTTAGAGACT769-15 161 ACAGGTTAGCATAATGTGCTAGTGCG 48  95.83 82.69CCCTCAAACCTATTTAGAGACT 769-16 162 ACAGGTTAGCATAATGTGCCAGTGCG 48  95.8382.69 CCTTCAAACCTATTTAGAGACT 769-17 163 ACAGGTCAGCATAATGGGCTAGTGC 48 97.92 84.62 GCCTTCAAACCTATTTAGAGACT 769 223 ACAGSKYAKCAWRATGKGCHAKTGC48 — — Consensus GCMYTCAAACMTATYTDSAGACT Sequence 795-1 164ACAGCGAAGCATAATGGCTACTGAC 49 100 83.02 GCCCTCAAACCCTATTTGCAGACT 795-2165 ACAGCGAAGCATAATGGCTACTGAC 49  97.96 81.13 GCCCTCAAACCCTATTTACAGACT795-3 166 ACAGCGAAGCATAATGGCTTCTGAC 49  97.96 81.13GCCCTCAAACCCTATTTGCAGACT 795-4 167 ACAGCCAAGCATACTGGCTACTGAC 49  95.9279.25 GCCCTCAAACCCTATTTGCAGACT 795-5 168 ACAGCGAAGCATAATGGCTACTGAC 49 97.96 81.13 GCCCGCAAACCCTATTTGCAGACT 795-6 169ACAGCGAAGCATAATGGCTACTGAC 49  97.96 80.77 GGCCTCAAACCCTATTTGCAGACT 795-7170 ACAGCGAGGCATAATGGCTACTGAC 49  97.96 81.13 GCCCTCAAACCCTATTTGCAGACT795-8 171 ACAGCGAAGCATAATGGCTACTGAC 49  97.96 84.91GCCTTCAAACCCTATTTGCAGACT 795-9 172 ACAGCGAAGCATAATGGCTACAGAC 49  95.9280.77 GCCCTCAAAACCTATTTGCAGACT 795-10 173 ACAGCGAAGCATAATGGCTACTGAC 48 97.96 83.02 GCCCTCAAACCCTATTTGAGACT 795-11 174ACAGCGAAGCATAATGGCTACTGAC 48  93.88 76.92 GCCCTCAAACCCTATTGTCGACT 795-12175 ACAGCCAAGCATAATGGCTACTGAC 49  97.96 81.13 GCCCTCAAACCCTATTTGCAGACT795-13 176 ACAGCGAAGCATAATGGCTACTGAC 49  95.92 83.02GCCCTCAAACCCTATTTGGCGACT 795-14 177 ACAGCGAAGCATAATGTCTACTGAC 49  97.9681.13 GCCCTCAAACCCTATTTGCAGACT 795-15 178 ACAGCGAAGCATAATGGCTACTGAC 49 95.92 83.02 GCCGTCAAACCCTATTTGTAGACT 795-16 179ACAGCGAAGCATAATGGCTACTGAC 49  97.96 83.02 GCCCTCAAACCTTATTTGCAGACT795-17 180 ACAGGTAGCATAATGGCTACTGACG 48  95.92 84.91CCCTCAAACCCTATTTGCAGACT 795-18 181 ACAGCGAAGCATAATGGCTACTGAC 49  95.9281.13 GCCCTCAAACCCTATTTCTAGACT 795-19 182 ACAGCGAAGCATAATGGCTACTGAC 49 97.96 83.02 GCCCTCAAACCCTATTTGTAGACT 795 224 ACAGNSWRGCATAMTGKCTWCWGA49 — — Consensus CGSCBKCAAAMCYTANTTVNMGACT SequenceWhere the N at position 5 can be C or nonucleotide, the N at position 40 can be T orno nucleotide, and the N at position 44 can be C, G, T, or no nucleotide935-1 183 ACAGGGTAGCATAATGGGCTACTTG 48 100 86.79 ACGCCTTCACCTATTTGTAGACT935-2 184 ACAGGGTAGCATAATGGGCTACTTG 47  97.92 86.79ACGCCTTCACCTATTTGAGACT 935-3 185 ACAGGGTAGCATAATGGGCTACTTTA 48  97.9284.62 CGCCTTCACCTATTTGTAGACT 935-4 186 ACAGGGTAGCATAATGGGCTACTTG 48 97.92 84.91 ACGCCTTCACCTATTTCTAGACT 935-5 187 ACAGGGTAGCATAATGGGCTACTTG48  97.92 88.68 ACGCCTTCACCTATTTGGAGACT 935-6 188ACAGGGTAGCATAGTGGGCTACTTG 48  97.92 84.91 ACGCCTTCACCTATTTGTAGACT 935-7189 ACAGGGTAGCATGATGGGCTACTTG 48  97.92 84.91 ACGCCTTCACCTATTTGTAGACT935-8 190 ACAGGGTAGCATAATGGGCTACTTG 48  97.92 84.91ACGCCTTCACCTATTAGTAGACT 935-9 191 ACAGGGTAGCATAATGGGCTATTTGA 48  97.9284.91 CGCCTTCACCTATTTGTAGACT 935-10 192 ACAGGGTAGCATAATGGGCTACTTGC 48 97.92 86.54 CGCCTTCACCTATTTGTAGACT 935-11 193ACAGTGTAGCATAATTGGCTACTTGA 48  95.83 83.02 CGCCTTCACCTATTTGTAGACT 935-12194 ACAGGGTAGCATAATGGGCTACTTG 48  95.83 83.02 ACGCTTTCACCTTTTTGTAGACT935-13 195 ACAGGGTAGCATAAGGGGCTACTTG 48  97.92 84.91ACGCCTTCACCTATTTGTAGACT 935-14 196 ACAGGGTAGCATAATGGACTACTTG 48  95.8381.13 ACGCCTCCACCTATTTGTAGACT 935-15 197 ACAGGGTAGCATAATGGGCTACTTGT 48 97.92 84.62 CGCCTTCACCTATTTGTAGACT 935 225 ACAGKGTCGCATRRKKGRCTAYTTKH48 — — Consensus CGCYTYCACCTWTTWSNAGACT SequenceWhere the N at position 43 can be G, T, or no nucleotide. 946-1 198ACAGCGTAGCATAATGGGCTGCAGA 49 100 84.62 CGCCGTCAAACCTATTTGCAGACT 946-2199 ACAGCGTAGCATAATGGGCTGCAGA 49  97.96 82.69 CGCAGTCAAACCTATTTGCAGACT946-3 200 ACATGTAGCATAATGGGCTACTGACG 48  91.84 86.54CCGTCAAACCTATTTGCAGACT 946-4 201 ACAGCGTAGCATAGTGGGCTGCAGA 49  97.9682.69 CGCCGTCAAACCTATTTGCAGACT 946-5 202 ACAGTGTAGCATAATGGGCTGCAGA 49 93.88 88.46 CGCCTTCAAACCTATTTGGAGACT 946-6 203ACAGTGTAGCATAATGGGCTGCTGAC 49  93.88 86.54 GCCGTCAAACCTATTTGAAGACT 946-7204 ACAGCGTAGCATAATGGGCTACAGG 49  95.92 84.62 CGCCGTCAAACCTATTTGCAGACT946-8 205 ACAGCGTAGCATAATGGGCTACTGG 49  93.88 86.54CGCCGTCAAACCTATTTGCAGACT 946-9 206 ACAGCGTAGCATAATGGGCTGCAGA 48  97.9684.62 CGCCGTCAAACCTATTTGAGACT 946-10 207 ACAGGTAGCATAATGGGCTGCAGAC 48 97.96 84.62 GCCGTCAAACCTATTTGCAGACT 946-11 208ACAGGTAGCATAATGGGCTGCTGAC 48  93.88 84.62 GCCGTCAAACCTATTTACAGACT 946-12209 ACAGCGTAGCATATTGGGCTGCAGA 49  97.96 82.69 CGCCGTCAAACCTATTTGCAGACT946-13 210 ACAGCGTAGCATAATGGGCTGCAGA 49  95.92 88.46CGCCTTCAAACCTATTTGGAGACT 946-14 211 ACAGTGTAGCATAATGGGCTGCAGA 48  95.9284.62 CGCCGTCAAACCTATTTGAGACT 946-15 212 ACAGCGTAGCATAATGGGCTGCTGA 49 95.92 88.46 CGCCGTCAAACCTATTTGGAGACT 946-16 213ACAGCGTAGCATAATGGGCTGCAGA 49  97.96 82.69 CGCCGTCAAACCTATTTACAGACT946-17 214 ACAGCGTAGCATAATGGGCTGCTGA 49  97.96 86.54CGCCGTCAAACCTATTTGCAGACT 946-18 215 ACAGGGTAGCATAATGGGCTGCAGA 49  95.9288.46 CGCCGTCAAACCTATTTGGAGACT 946-19 216 ACAGCGTAGCATAATGGGCTACAGA 49 97.96 86.54 CGCCGTCAAACCTATTTGCAGACT 946-20 217ACAGCGTCGCATAATGGGCTGCAGA 49  95.92 80.77 CGCCGTCAAATCTATTTGCAGACT946-21 218 ACAGCGTAGCATAATGGGCTTCAGA 49  97.96 84.62CGCCGTCAAACCTATTTGCAGACT 946-22 219 ACATGTAGCATAATGGGCTGCAGAC 48  93.8884.62 GCCGTCAAACCTATTTGGAGACT 946 226 ACANNGTMGCATADTGGGCTDCWGR 49 — —Consensus CGCMKTCAAAYCTATTTRNAGACT SequenceWhere the N at position 4 can be G or nonucleotide, the N at position 5 can be C, G,T, or no nucleotide, and the N at position 44can be A, C, G, or no nucleotide. 961-1 220 ACACCGTAGCATAATGGGCTACTGCC47 100% 82.69 GCCGTCGACCTTTTGGAGACT 996-1 221 ACAGGGTAGCATAATGGCTTAGGAC46 100% 76.92 GCCTTCAAACCTATCAAGACT Candidates for binding acyclovir andpenciclovir. DNA sequences corresponding to the non-stem regions of theacyclovir binding RNA riboswitches. Seven families were identified inthe screen: 582, 769, 795, 935, 946, 961, and 996 with between 1 and 39sequences in each family. The percent identity for each sequence in thefamily was compared to the most prevalent sequence within each family(582-1, 769-1, 795-1, 935-1, 946-1, 961-1, and 996-1). The percentidentity for each sequence in the family was also compared to thewild-type sequence. tab

Positive target acyclovir produced seven strong candidates (SEQ IDNOs:87-93; RNA sequences including stem regions) corresponding to 582-1(SEQ ID NO:108), 769-1 (SEQ ID NO:147), 795-1 (SEQ ID NO:164), 935-1(SEQ ID NO:183), 946-1 (SEQ ID NO:198), 961-1 (SEQ ID NO:220), and 996-1(SEQ ID NO:221), each designated F1A (FIG. 17 ). These sequences werethe most prevalent sequences in each family (the DNA sequences of allthe members of each family are: 582 (SEQ ID NOs:108-146); 769 (SEQ IDNOs:147-163); 795 (SEQ ID NOs:164-182); 935 (SEQ ID NOs:183-197); 946(SEQ ID NOs:198-219); 961 (SEQ ID NO:220); and 996 (SEQ ID NO:221)). Theconsensus sequences show all possible substitutions or gaps at eachnucleotide position for each family (SEQ ID NOs:222-226). As the goalwas to identify aptamers from a library based on RNA that is known tobind to deoxyguanosine, strong candidates needed to have minimalpresence in the counter-targets population. Candidates F1A-795, F1A-935,and F1A-946 met this criterion very well, as they were not detected inthe counter-target population. F1A-996 and F1A-961 are considered thenext best candidates in this regard, although they do show up to a smalldegree in the counter-targets population. In addition, candidates shouldappear minimally in the negative population, as those sequences desorbedfrom GrO without the influence of acyclovir and could represent falsepositives. F1A-935 and F1A-946 performed ideally under this criterion aswell, as they were not found in the negative population. CandidateF1A-769 was minimally detected in the negative population, withcandidates F1A-961, F1A-795 and F1A-996 performing less well. Enrichmentrate was the final condition to be considered, with F1A-935, F1A-946,and F1A-769 performing adequately. Candidate F1A-582 was includedbecause it exhibited the greatest enrichment rate, although it did notperform well under the other criteria. The remaining candidates did notperform well relative to these four, but exhibited acceptablecharacteristics.

Additional target penciclovir produced seven strong candidates (SEQ IDNOs:94-100), each designated F1P (FIG. 18 ). As before, the goal was toidentify aptamers from a library based on RNA that is known to bind todeoxyguanosine, diverging from libraries enriched for binding toacyclovir (acyclovir) after Round 10. Strong candidates needed to haveminimal presence in both the acyclovir and the counter-targetspopulations to minimize cross-reactivity. Candidate F1P-923 met thefirst criterion, candidate F1P-710 met the second criterion, andcandidate F1P-584 met both criteria to a degree. Candidate F1P-584 alsodemonstrated moderate favorability for penciclovir over the negativecondition, as well as moderate enrichment relative to the previousgeneration's response to acyclovir. The remaining candidatesdemonstrated either minimal favoring of penciclovir over acyclovir orminimal favoring of penciclovir over counter-targets (F1P-837 andF1P-932; F1P-991 and F1P-718; respectively). These four candidatesdemonstrated some favorability for penciclovir over the negativecondition which minimizes the chance of a false positive, although thiscriterion is not as significant if a candidate does not demonstrateselectivity for penciclovir over its analogues. Enrichment rate was thefinal condition to be considered, with F1P-923, F1P-932, and F1P-584performing adequately.

Qualitative PAGE assessment of selected aptamers was performed.Individually synthesized and transcribed aptamers were subjected toselection on Graphene Oxide (GrO) under physiological Mg++ (0.5 mM) andelution with either acyclovir (+) or counter-targets (x). Thespecifically eluted aptamer fractions for each sample were subjected toPAGE for analysis.

100 pmoles of each aptamer candidate (per trial/lane) was resuspended in1× modified selection buffer (50 mM HEPES, 100 mM KCl, 0.5 mM MgCl₂, pH7.3) and refolded (90° C. for 5 min, then 4° C. for 5 min), thenincubated at 37° C. for 30 minutes with 200 pmoles (each) of pooledcounter-targets or target. Final library concentration was 0.5 μM,target/counter-targets concentration was 1 μM (incubation volume was 200l).

After target/counter-target incubation, 250 μg of GrO (AngstronMaterials (Dayton, OH) was added to adsorb unbound candidate (10-minuteincubation at 37° C.).

Samples were centrifuged for 5 minutes at 7,000×g. Supernatant wasrecovered, denatured using 2× Formamide with 40 mM EDTA, and run on 10%denaturing PAGE with 8 M urea (supplier: American Bioanalytical; catalog#'s AB13021-01000. AB13022-01000). Running buffer was 1×TBE (supplier:Amresco/VWR; catalog #0658-20L, diluted using DI water). DNA ladder was20/100 DNA ladder (IDT). Gels stained with Gel Star (Lonza, 50535) andimaged on a blue light transilluminator.

Candidates F1A-769, F1A-795, F1A-946, and F1A-996 appear to exhibitselective positive response in this qualitative PAGE assessment (goodelution of the Aptamer from GrO with Acyclovir target and relativelylower or minimal elution with counter-targets).

Conclusion

Strong candidates for acyclovir were identified after twelve rounds ofiterative screening and parallel assessment; reasonable candidates forpenciclovir were identified after two rounds of screening and parallelassessment.

Example 2. Isolation of Conditional scFv's

Potential splice site liabilities are removed and tumor antigen specificscFv's are synthesized by overlapping oligo synthesis and cloned intothe CAR shuttle construct containing the acyclovir responsive elementand the primate CD3ζ promoter. As an initial prototype, anti-ECD ofEPCAM or ERBB2scFv with a CD8-alpha signal peptide, stalk, andtransmembrane domain is utilized. Solid tumor microenvironmentrestricted CAR products are generated either using methods as describedin U.S. Pat. No. 8,709,755 and PCT Publication No. WO/2016/033331A1 orby direct selection from human phage libraries under permissive andnon-permissive conditions. Briefly, a human V_(H)×V_(L) library fromCreative Biolabs (Shirley, NY) is panned in the following tumorpermissive conditions: 100 μg/ml hyaluronan, 100 kDa fraction (LifecoreBiomedical, Chaska, MN), 20 mg/ml recombinant HSA (Cyagen, Santa Clara,CA), 200 ng/ml recombinant human VEGF in 25 mM sodium bicarbonatebuffer, 2 μM adenosine, 10 mM sodium lactate pH 6.7, following clearancewith streptavidin magnetic beads (ThermoFisher, Carlsbad, CA) bound tobiotinylated human IgG. Binding to biotinylated-target receptor ECD ofEPCAM and ERBB2 conjugated beads at 37° C. is performed under permissiveconditions followed by serial washes in permissive conditions. Phage arereleased with physiologic conditions (1 μg/ml hyaluronan, 20 mg/ml HSA,25 mM bicarbonate, 1 mM sodium lactate pH 7.2) followed by elution oftight variants with acid elution and rapid neutralization with 1 M Tris.Phage are expanded and genomic DNA is split for deep sequence analysisof V_(H)×V_(L) chains using long read sequencing (PacBio, Menlo Park,CA). Panning can be repeated for enrichment. V_(H)×V_(L) sequencesshowing preferential amplification of reads during the phage culturingprocess over enrichment to target are excluded for further analysis.Phage with selective binding to the target that are enriched under tumorpermissive conditions but released under physiologic conditions arechosen for further characterization by cloning into the CAR constructexpression system, generation of lentivirus, and transduction into Tcells for testing CAR-mediated tumor antigen expressing target cellkilling in a tumor-selective environment compared to physiologicconditions.

Example 3. Generation of MRB-Cars Using Microenvironment RestrictedscFv's

Microenvironment restricted ASTRs were obtained that were made bysubjecting V_(H) and V_(L) sequences with low selectivity for the low pHmicroenvironment by evolution as described in applicationWO/2016/033331A1. Chimeric antigen receptors (CARs) for binding eitherof two cognate tumor antigens, Target 1 or Target 2, with increasedactivity at the reduced pH of a tumor microenvironment compared to themicroenvironment of normal tissue MRB-CARs were made by incorporatingthe heavy chains and light chains of the microenvironment restrictedsingle-chain antibodies into lentiviral expression vectors along withother CAR domains to generate a series of candidate MRB-CARs. TheseMRB-CARs included various combinations of modules. The MRB-CARsincluded, from amino to carboxy terminus, in positions 1 through 9, aCD8 signal peptide (sp) (P1) (SEQ ID NO:74); a microenvironmentrestricted anti-Target 1 ASTR or anti-Target 2 ASTR (P2-P4); a stalk andtransmembrane (TM) domain from CD8 (SEQ ID NO:75) (P5) and aco-stimulatory domain from CD137 (P6) (SEQ ID NO:1) in the cases of T2Aand T2B or a stalk and transmembrane (TM) domain from CD28 (SEQ IDNO:76) (P5) and a co-stimulatory domain from ICA (SEQ ID NO:3) (P6) inthe case of TIA; an activation domain from CD3Z (SEQ ID NO:13) (P7); a2A-1 ribosomal skip sequence (SEQ ID NO:77) (P8); and an exemplary eTAG(SEQ ID NO:78) (P9).

Pan T cells (AllCells, Alameda, CA) were transduced with the recombinantlentiviral particles to express the series of candidate MRB-CARs and thepercent transduced cells was calculated by determining the percent ofcells expressing the eTag using FACS. Pan T cells were successfullytransduced with the recombinant lentiviral particles encoding thecandidate MRB-CARs.

The cytotoxic activity of the candidate MRB-CARs against target cellsexpressing either Target 1 or Target 2 (CHO-Target 1 and CHO-Target 2,respectively) was analyzed at a pH of 7.4 (normal tissue) or a pH of 6.7(reduced pH of a tumor microenvironment) by xCELLigence System (ACEA).Briefly, target cells expressing Target 1 or Target 2 were seeded to a96-well E-plate at 20,000 cells/well with tumor conditional or normalmedium one day before the experiment. Effector cells were rested for twodays in human T cell medium containing 100 IU/mL of IL-2 and added intoexperimental wells containing Target cells at effector cell/target cellratios (E/T) of 3:1, 1:1, and 0.3:1.

Impedance readings were taken every 5 minutes for approximately 40 hoursafter effector cell addition and impedance was reported as the CellIndex (CI). Percentage of specific cytolysis was calculated as follows((CI Target+Control virus transduced effector T cells)−(CITarget+effector T cells transduced with CARs directed to Target 1 orTarget 2))/(CI Target+Control virus transduced effector T cells)×100.

Results

Many of the candidate MRB-CARs had higher cytotoxic activity on thetarget cells at a pH of 6.7 than at a pH of 7.4. Exemplary MRB-CARs thatwere more effective at lysing target cells at a pH of 6.7 than at a pHof 7.4 included MRB-CAR T1A, MRB-CAR T2A, and MRB-CAR T2B. The ASTR ofMRB-CAR T1A comprised, from 5′ to 3′, Target 1 MRB VH (SEQ ID NO:281)and Target 1 MRB VL (SEQ ID NO:282) separated by Linker 1 (SEQ IDNO:283). The ASTR of MRB-CAR T2A comprised, from 5′ to 3′, Target 2 MRBVH (SEQ ID NO:289) and Target 2 MRB VL (SEQ ID NO:290) separated byLinker 2 (SEQ ID NO:291). The ASTR of MRB-CAR T2B was the same as thatfor MRB-CAR T2A except that the positions of VH and VL were swapped.

Example 4. Construction of Ligand-Inducible Riboswitches

Deoxyguanosine riboswitch aptamer and guanine riboswitch aptamers(Pikovskaya, 2014; Kim, 2007) or other purine riboswitch aptamers aresynthesized as oligonucleotides. In one example, the deoxyguanosine IAriboswitch from Mesoplasma florum (underlined and in bold in FIG. 6 ;FIG. 7 ) is selected for evolution to generate an acyclovir-responsiveriboswitch. In another example, the guanine xpt riboswitch from Bacillussubtilis (underlined and in bold in FIG. 10 ; FIG. 11 ) is selected forevolution to generate an acyclovir-responsive riboswitch. For each ofthese two examples, a random RNA library is generated with alternatenucleotides at targeted sequence positions in the P2, P3, J1-2, and J2-3segments (FIGS. 7 and 11 ). Each segment allows for 3 alternate nucleicacids at each targeted sequence position, or alternatively base deletionand insertion of 4 nucleotides in the +1 site at each targeted sequenceposition for saturation mutagenesis as indicated in FIGS. 8A-8B and 9(M. florum IA) and FIGS. 12A-12B and 13 (B. subtilis xpt). Primerextension and reagent preparation is followed by RNA transcription. Theresultant RNA library is negatively selected on graphene oxide in thepresence of guanine, guanosine, and deoxyguanosine followed by positiveselection with acyclovir or penciclovir. During the negative andpositive selection processes, human cell physiologic magnesium levels(0.5 mM to 1.2 mM) are used and the temperature is kept at 37° C.Recovered aptamers are reverse transcribed and PCR amplified followed bytranscription and subsequent screening for at least 8 successive roundsof selection. In a parallel approach, aptamers are screened with anadditional negative screen at 40° C. Resultant positive pools areexamined by NextGen sequencing and analysis. Individual aptamers aresynthesized and examined for affinity by isothermal calorimetry at35-40° C. in human cell physiologic magnesium levels. Followingselection for positive acyclovir and penciclovir specific aptamers,aptamers are integrated with ribozyme hammerhead and pistol ribozymes.Positive acyclovir selective aptamers are combined with pistol ribozymesto identify acyclovir regulated ribozymes. (Harris KA RNA. 2015 Nov.;21(11):1852-8. doi: 10.1261/rna). Variants are subjected to gel shiftbased PAGE purification in the presence of acyclovir and absence ofpenciclovir. Additionally, the acycloguanosine selective riboswitch isplaced immediately 3′ in a loop to a splice acceptor upstream of theCAR/IL-7 construct. In the absence of acyclovir, the splice siteposition is bound in the riboswitch complex but in the presence ofacyclovir becomes accessible, generating a functional CAR transcript.

Example 5. Construction of In Vivo Propagation Domains

A series of constitutively active IL7 receptor (IL7R) transmembranemutants from T cell lymphoblastic leukemias (243 InsPPCL (SEQ ID NO:82);246 InsKCH (SEQ ID NO:101); 241 InsFSCGP (SEQ ID NO:102); 244 InsCHL(SEQ ID NO:103); and 244 InsPPVCSVT (SEQ ID NO:104); all from Shochat etal 2011, J. Exp. Med. Vol. 208 No. 5 901-908) are synthesized byoverlapping oligo nucleotide synthesis (DNA2.0, Newark, California). Thesynthesized constitutively active IL7R transmembrane mutants areinserted into a constitutively expressing lentiviral vector backboneimmediately behind a 2A ribosomal skip sequence followed by an anti-CD19CD3ζ expression cassette, which includes a CD8A stalk (SEQ ID NO:79) anda leader peptide (SEQ ID NO:74). HEK293 packaging cells are transfectedwith the IL7R transmembrane mutant lentiviral vectors and lentiviralpackaging constructs, grown, and viral supernatants are harvested usingmethods known in the art. CD3/CD28-stimulated T cells are transducedwith the viral supernatants and grown in IL2 deficient AIM V, CTSOpTmizer T Cell Expansion SFM, or X-VIVO 15 media for 4 weeks,supplemented weekly with frozen PBMCs from the same donor. The resultingexpanded transduced T cells expressing IL7R variants are cloned by FACSsorting and the sequences of the IL7R constructs are identified bysequencing RT-PCR products. The 243 InsPPCL variant (PPCL) (SEQ IDNO:82) is selected for further evolution to generate a conditionallyactive CAR.

Example 6. Screening of Accessory Components for CAR-T Activation andPropagation

A series of protein-encoding domains (ABCG1, SOCS1, SMAD2, TGFBR2, cCBL,and PD1) and miRNA sequences are constructed for incorporation into asynthetic intron on the reverse strand of a CD3-promoter driven CARcassette. Each construct containing the CD3-promoter driven CAR cassetteand a protein-encoding domain or miRNA sequence includes a unique barcode for deep sequencing and is assembled using Gibson assembly followedby transformation and library expansion in E. coli. Viral stocks areproduced and used to transduce CD3/CD28-stimulated T cells in AIM V, CTSOpTmizer T Cell Expansion SFM, or X-VIVO 15 media without IL2 andallowed to grow for 4 weeks in culture with serial sampling of DNA foramplification and deep sequencing for code identification. The libraryis also subject to PACBio full length sequencing to determine librarydiversity and to decode the bar code components. The miRNA sequences andprotein-encoding domains are tested for synergistic activation of CARCD3ζ domains.

Example 7. Engineering a Retroviral Packaging and Transducing System toTarget Resting T Cells for Selective T Cell Integration and Expressionfrom PBMCs

Although producing high-titer lentiviral vectors by transienttransfection is possible, this method carries the risk of generatingreplication competent retroviruses (RCRs) and is not scalable forclinical applications. Herein, a stable retroviral packaging cell lineis generated by the simultaneous introduction of multiple constructsencoding inducible promoters and their regulators into HEK293suspension-adapted cells (HEK293S) to stably produce the viralcomponents, CAR genes, and their regulatory components. Two distinctinducible systems can be used to temporally control the expression ofgenes. One system is based on rapamycin- or rapalog-induced dimerizationof two transcription factors. One transcription factor consists of threecopies of the FKPB protein fused to a ZFHD1 DNA binding domain and theother transcription factor consists of a FRB protein fused to a p65activation domain. Rapamycin or a rapalog dimerizes the transcriptionfactors to form ZFHD1/p65 AD and can activate gene transcription at12×ZFHD1 binding sites.

A series of vectors as shown in FIGS. 3A-3E are generated with flankingtransposon sequences for integration into the HEK293S genome. Onceintegrated into the genome of a cell, these sequences function asregulatory components and lox and/or FRT sites for subsequentintegration using Cre and/or flp recombinases, herein referred to aslanding pads. The initial 5 constructs contain polynucleotide sequencesencoding puromycin resistance, GFP, RFP, and an extracellular MYC tagthat is targeted to the cell membrane through an N-terminal PLss (bovineprolactin signal peptide) and anchored to the cell membrane through aplatelet-derived growth factor receptor (PDGFR) C-terminal transmembraneanchoring domain. The initial 5 constructs can also include constitutiveminimal CMV and minimal IL-2 promoters, a rapamycin-regulatedZFHD1-based promoter, a tetracycline-responsive element (TRE) promoter,or a bidirectional TRE (BiTRE) promoter. The construct in FIG. 3Acontains a polynucleotide sequence encoding FRB domain fused to the NFκBp65 activator domain (p65 AD) and ZFHD1 DNA binding domain fused tothree FKBP repeats that is constitutively expressed. The construct inFIG. 3A also includes HIV1 REV and HSV VP65 domain SrcFlagVpx under therapamycin-inducible ZFHD1/p65 AD promoter. The construct in FIG. 3Bincludes a polynucleotide encoding an rtTA sequence under the control ofthe ZFHD1/p65 AD promoter. The construct in FIG. 3C includes apolynucleotide encoding a puromycin resistance gene flanked by loxPsites and the extracellular MYC tag flanked by lox2272 sites. Both ofthese selectable markers are under the control of a BiTRE promoter,which is flanked by FRT sites. The construct in FIG. 3D includes apolynucleotide encoding GFP flanked by loxP sites that is under thecontrol of a TRE promoter. The construct in FIG. 3D also includes asingle FRT site between the TRE promoter and the 5′ loxP site of GFP.The construct in FIG. 3E includes a polynucleotide encoding RFP flankedby loxP sites that is under the control of the ZFHD1/p65 AD promoter.The construct in FIG. 3E also includes a single FRT site between theZFHD1/p65 AD promoter and the 5′ loxP site of RFP The constructs inFIGS. 3C-3E function as landing pads for other polynucleotide sequencesto insert into the genome of the packaging cell line. The polynucleotidesequences to be inserted can be flanked by lox sites and inserted intothe genome using Cre recombinase and the loxP sites. This results ininsertion and simultaneous removal of the genomic regions encodingpuromycin resistance, the extracellular MYC tag, GFP, and RFP.Alternatively, the polynucleotide sequences can be flanked by FRT sitesand inserted into the genome using flp recombinase and the FRT sitesfollowed by removal of the polynucleotide sequences encoding puromycinresistance, the extracellular MYC tag, GFP, and RFP using Crerecombinase.

To generate the packaging cell line with landing pads integrated intothe genome, HEK293S cells are co-transfected with equimolarconcentrations of the 5 plasmids (FIGS. 3A-3E) plus 5 μg of invitro-transcribed piggybac transposase mRNA or 5 μg of a plasmid with apromoter for expressing piggybac transposase in the presence of PEI at aratio of 2:1 or 3:1 PEI to DNA (w/w) or 2-5 μg piggybac transposaseprotein using a cationic peptide mixture. The transfected cells areselected with puromycin in the presence of 100 nm rapamycin and 1 μg/mLdoxycycline for 2-5 days followed by fluorescence-activated cell sortingto collect cells expressing GFP and RFP. The sorted cells are grown 5days in the absence of puromycin, rapamycin, and doxycycline and cellsexpressing GFP and RFP are removed also myc positive cells are removedwith myc beads. Individual clones from negatively sorted cells are thenscreened for induction of GFP and RFP by rapamycin and doxycycline andsingle cell cloned. The DNA from clones is harvested and sequenced forintegration analysis. Clones positive for strong inducible expression ofGFP and RFP in the presence of rapamycin and doxycycline with limitedbackground expression in the absence of rapamycin and doxycycline areexpanded and banked.

The HEK293S cells with the constructs from FIGS. 3A-3E integrated intothe genome are then transfected with a construct containing atricistronic polynucleotide encoding a DAF signal sequence/anti-CD3scFvFc (UCHT1)/CD14 GPI anchor attachment site (SEQ ID NO:287), a DAFsignal sequence/CD80 extra-cellular domain capable of binding CD28/CD16BGPI anchor attachment site (SEQ ID NO:286), and a DAF signalsequence/IL-7/DAF (SEQ ID NO:286) and transposon sequences flanking thepolynucleotide region for integration into the HEK293S genome (FIG. 4A).After transfection, cells are expanded for 2 days in the absence ofrapamycin and doxycycline and colonies that are constitutively red areselected. Positive colonies are then transiently transfected with aconstruct for expressing Cre recombinase to remove remaining genomicDNA, and the RFP encoding region. Another construct (FIG. 4B) containinga polynucleotide with a BiTRE promoter and a polynucleotide regionencoding the gag and pol polypeptides in one direction and apolynucleotide region encoding the measles virus F and H proteins in theother direction is transfected at the same time. The Cre recombinaseintegrates the construct into the genome to generate the integratedsequence shown in FIG. 4B. Resultant colonies are evaluated for proteinexpression in the presence of doxycycline and rapamycin and analyzed bydeep sequencing for genomic integration. The remaining TRE responsiveGFP site is retained for the lentiviral genome insertion.

Example 8. Generation of Lentivirus Vector and Retroviral Packaging

The retroviral packaging stable cell line generated in Example 7 istransfected with a construct (FIG. 4C) for expressing Flp recombinaseand a construct containing a polynucleotide sequence encoding a CAR andthe lymphoproliferative element IL7Rα-insPPCL under the control of aCD3Z promoter that is not active in HEK293S cells, wherein the CAR andIL7Rα-insPPCL are separated by a polynucleotide sequence encoding a T2Aribosomal skip sequence and the IL7Rα-insPPCL has an acyclovirriboswitch controlled ribozyme. The CAR-containing construct furtherincludes cPPT/CTS and RRE sequences and a polynucleotide sequenceencoding HIV-1 Psi. The entire polynucleotide sequence on theCAR-containing construct to be integrated into the genome is flanked byFRT sites. Successful integration of the CAR-containing construct causesconstitutive expression of GFP that is consequently removed by transienttransfection with a construct for expressing Cre recombinase. TheHEK293S line is grown in serum free media. Following growth to peak celldensity in a stirred tank reactor, the cells are diluted to 70% peakcell density and treated with 100 nM rapamycin for 2 days to induceexpression of early genes REV, Vpx, and aCD3 scFv CD16B GPI, aCD28 scFvCD16B GPI, and IL-7 SD GPI DAF followed by the addition of 1 ug/mLdoxycycline in the media to induce expression of structural elementslike Gag Pol, MV(Ed)-FA30, MV(Ed)-HA18, and lentiviral genome includingthe therapeutic target. Levels of virus production are examined by qPCRof the packaging sequence and p24 ELISA. Virus is harvested by depthfiltration of cells, and concentration/diafiltration using a TFFcartridge followed by flash freezing for vialing.

Example 9. Peripheral Blood Mononuclear Cell (PBMC) Isolation,Transduction, and Expansion

The following example illustrates the use of a closed system for ex vivoprocessing of PBMCs before in vivo expansion. As an example, 30 to 200ml of human blood is drawn from a subject with Acid Citrate DextroseSolution (ACD) as an anticoagulant into a blood collection bag.Alternatively, blood is drawn into Vacutainer tubes, a syringe, or anequivalent and is transferred to an empty blood collection or IV bag.The whole blood is processed using a Neat Cell kit (Cat #CS-900.2,Omniamed) on a Sepax 2 cell processing system (BioSafe) according to themanufacturers' instructions. The peripheral blood mononuclear cells(PBMCs) are collected either into a culture bag, or alternatively asyringe. An aliquot is taken aseptically for cell counting to determinethe number of viable cells. The PBMCs are transferred to a G-Rex100MCSGas Permeable Cell Culture System device (Wilson Wolf) at a finalconcentration of 0.1-1.0×10⁶ viable cells/ml in X-VIVO 15 (Cat #08-879H,Lonza) or CTS OpTmizer Cell Expansion SFM (Cat #A1048501, Thermo FisherScientific) media with 10-300 IU/ml IL-2 (Cat #202-IL-010, R&D Systems)in up to 200 ml final volume. In addition to IL-2, CTS Immune Cell SR(Cat #A2596101, Thermo Fisher Scientific) can be added to the media. Theclosed G-Rex Gas Permeable Cell Culture System device can be pre-coatedwith Retronectin (Cat #CH-296, Takara), or a similar fibronectin-derivedequivalent, according to the manufacturer's instructions.

The PBMCs isolated from peripheral blood are loaded onto a PALL PBMCfilter, washed once through the filter with 10 ml of AIM V (ThermoFisher Scientific) or X-VIVO 15 media followed by perfusion with 10-60ml of lentivirus stock (as prepared in Example 8) at 37° C. at 5 ml/hr.The PBMCs are then washed again with AIM V, CTS OpTmizer T CellExpansion SFM, or X-VIVO 15 media containing recombinant human DNase(Pulmozyme, Genentech) followed by a wash with DNase-free LactatedRingers (Cat #L7500, Braun). The PBMCs are then reverse perfused throughthe filter into a syringe. The cells (target levels of cells are 5×10⁵to 1×10⁶ cells/kg) are then reinfused into the subject throughintravenous infusion.

Depending upon the riboswitch contained within the retroviral genome,the subject is given the respective nucleoside analogue antiviral drugor nucleoside analogue antiviral prodrug (acyclovir, valaciclovir,penciclovir, or famciclovir). Subjects can be given any therapeuticallyeffective dose, such as 500 mg of the nucleoside analogue antiviral drugor prodrug orally three times/day. Treatment with the nucleosideanalogue antiviral drug or prodrug preferably begins before reinfusion,such as 2 hours before, and can also begin at the time of reinfusion orat some time after reinfusion. The treatment can continue for at least1, 2, 3, 4, 5, 7, 10, 14, 21, 28, 30, 60, 90, 120 days or 5, 6, 9, 12,24, 36, or 48 months or longer. The treatment can include administrationof the nucleoside analogue antiviral drug or prodrug once, twice, three,or four times daily. After reinfusion and treatment is begun, the numberof infected cells is determined through blood counts on days 2, 5, 7,11, 13, 18, 28, and 56 post-reinfusion using qPCR to quantitate theamount of viral genome. A subject experiencing fever or cytokine releasesyndrome may have the dose or frequency of the nucleoside analogueantiviral drug or prodrug reduced or halted. If the infected T cellsfail to amplify 10,000-100,000 fold by day 18, the dose or frequency ofthe nucleoside analogue antiviral drug or prodrug may be increased. Theclinical response of the subject can be measured through FDG PET imagingand serial CT scan. Oral dosing of the nucleoside analogue antiviraldrug or prodrug can be reduced or halted following prolonged remissionor in the event of excessive T cell propagation beyond 30% of totalperipheral T cell counts.

Example 10. Therapeutic Intervention to Raise Vascular or Tissue pH

To reduce the binding of an antigen binding domain to its cognateantigen, NaHCO₃ is administered as an IV bolus or by IV infusion. Thestandard dosage is 1 mg/kg of body weight as the initial dose followedby 0.5 mg/kg every 10 minutes. A 50-milliliter bolus of NaHCO₃ willraise the serum pH approximately 0.1 of a pH unit. If the pH is 7.0, itrequires four 50 mEq ampules of HCO₃ to correct the pH to 7.40

Example 11. Testing Activity of IL-7 ReceptorLymphoproliferative/Survival Elements in PBMCs

To test IL-7Rα variants for their ability to mediatecytokine-independent survival of T cells, thirty milliliters of humanblood were drawn with acid citrate dextrose (ACD) as an anticoagulantinto Vacutainer tubes. The whole blood was processed using densitygradient centrifugation with Ficoll-Pacque™ (General Electric) followingmanufacturer's instruction, to obtain peripheral blood mononuclear cells(PBMCs). Aliquots of the PBMCs were transferred aseptically to wells ofa 12 well tissue culture plate, along with X-Vivo™ 15 media (Lonza) to afinal concentration of 0.5 million viable cells/mL in a final volume of1 mL. Recombinant human interleukin-2 (IL-2) (Novoprotein) was alsoadded to a concentration of 100 IU/ml in some samples. Activatinganti-CD3 Ab (OKT3, Novoprotein) was added at a concentration of 50ng/ml, to activate the PBMC for viral transduction. The plates wereincubated overnight in a standard humidified tissue culture incubator at37 degrees C. and 5% Carbon Dioxide. After overnight incubation,lentivirus particle preparations containing the desired test constructs(FIG. 19A) were added to individual wells at a multiplicity of infection(MOI) of 5. The plate was incubated overnight in a standard humidifiedtissue culture incubator at 37 degrees C. and 5% Carbon Dioxide.Following the overnight incubation, the contents of each of the wells ofthe 12 well plate were collected and centrifuged to obtain a pellet. Thesamples were washed once with D-PBS+2% Human Serum Albumin (HSA),resuspended in X-Vivo15™ media, and transferred to wells of G-Rex®6-well gas permeable cell culture devices (Wilson Wolf). AdditionalX-Vivo™ 15 media was added to bring the final volume of each well to 30ml. Matching control samples for each of the constructs were transferredto wells of G-Rex® 6-well gas permeable cell culture devices (WilsonWolf) and additional media was added to bring the final volume to 30 mlwith 100 IU/ml IL-2 for some control samples. The G-Rex® device wasincubated in a standard humidified tissue culture incubator at 37degrees C. and 5% Carbon Dioxide for 7 days. Fresh IL-2 was added to thecontrol samples containing IL-2 during the culture every 2-3 days.Matched test samples without IL-2 were not supplemented. Samples wereremoved for tracking cell numbers and viability during expansion(Countess, Thermo Fisher) at day 7.

FIG. 19A provides a schematic of the IL7Rα constructs that were tested.These constructs were inserted into a recombinant lentiviral genome. Thereplication incompetent recombinant retroviral particles were used totransduce PBMCs. FIG. 19A shows a schematic of wild-type IL7Rα (SEQ IDNO:229), which consists of a signal sequence (SS), an extracellulardomain (ECD), a transmembrane (TM), and an intracellular domain (ICD).“1” indicates the site of a fibronectin type III domain; “2″ indicatesthe site of a WSXWS motif”; “3” indicates a Box 1 site, “4” indicatesthe site of a protein kinase C (PKC) phosphorylation site, and “5”indicates a Box 2 site.

Variant “A” is the IL-7Rα with an InsPPCL at position 243 (Shochat et al2011, J. Exp. Med. Vol. 208 No. 5 901-908) but without the S185Cmutation, expressed on a transcript with a GFP polypeptide, a GSGlinker, and a P2A ribosomal skip sequence fused to its N-terminus.Variant “B” is the IL-7Rα InsPPCL with a GFP polypeptide, a GSG linker,and a P2A ribosomal skip sequence fused to its N-terminus as well as aMyc Tag between the signal sequence and the extracellular domain.Variant “C” is similar to variant “B” except its intracellular domain istruncated at position 292. Variant “D” is similar to variant “A” exceptits intracellular domain is truncated at position 292. Variant “E” isthe IL-7Rα InsPPCL variant truncated at its N terminus such that thesignal sequence and most of the extracellular domain (residues 1-228)are not present; variant “E” also has a GFP polypeptide, a GSG linker, aP2A ribosomal skip sequence, and an eTag fused to the N terminus, inthat order from the amino terminus. Numbering of the amino acid residuesis based on IL7Rα (NCBI GI No. 002176.2). T cells containing each of thevariants were tested for viability in the presence or absence of IL-2using Trypan Blue exclusion.

As shown in FIG. 19B, PBMCs require IL-2 for survival in vitro. Asillustrated in FIG. 19B, untransfected PBMCs have about 80% viability inthe presence of IL-2 and 0% viability in the absence of IL-2 at 7 daysafter transduction. PBMCs having the full-length versions of IL-7RαInsPPCL (IL-7Rα variants A and B in FIG. 19A) had over 20% viability inthe absence of IL-2 7 days after transduction, indicating thatexpression of the constitutively active IL-7Rα InsPPCL receptor hassurvival activity in these cells. Furthermore, T cells expressing theIL-7Rα InsPPCL variants with a truncated intracellular domain (ICD)(IL-7Rα variants C and D in FIG. 19A) had increased viability at 7 daysafter transduction compared to the wild-type IL-7 receptor. Finally, theN-terminal IL-7 receptor mutant (IL-7Rα variant E in FIG. 19A) as shownin FIG. 19B had survival activity in these cells. Accordingly, thisexample illustrates that IL-7 receptor has survival activity whenexpressed in PBMCs.

Example 12. Transduction Efficiency of Freshly Isolated and UnstimulatedHuman T Cells by Retroviral Particles Pseudotyped with VSV-G andExpressing Anti-CD3 scFvFc on their Surfaces

Recombinant lentiviral particles were produced by transient transfectionof 293T cells (Lenti-X™ 293T, Clontech) with separate lentiviralpackaging plasmids encoding gag/pol and rev, and a pseudotyping plasmidencoding VSV-G. A third generation lentiviral expression vector(containing a deletion in the 3′LTR leading to self-inactivation)encoding GFP, an anti-CD19 chimeric antigen receptor, and an eTAGreferred to herein as F1-0-03 (FIG. 20 ) was co-transfected with thepackaging plasmids. The cells were adapted to suspension culture byserial growth in Freestyle™ 293 Expression Medium (ThermoFisherScientific). The cells in suspension were seeded at 1×10⁶ cells/mL (30mL) in a 125 mL Erlenmeyer flask, and immediately transfected usingpolyethylenimine (PEI) (Polysciences) dissolved in weak acid.

Plasmid DNA was diluted in 1.5 ml Gibco™ Opti-MEM™ media for 30 mL ofcells. To obtain lentiviral particles pseudotyped with VSV-G, the totalDNA (1 μg/mL of culture volume) used was a mixture of 4 plasmids withthe following molar ratios: 2x genomic plasmid (F1-0-03), 1×Rev-containing plasmid, 1× VSV-G-containing plasmid, and 1×gag/pol-containing plasmid. To obtain lentiviral particles pseudotypedwith VSV-G and expressing an antiCD3-scFvFc-GPI on their surfaces, thetotal DNA (1 μg/mL of culture volume) used was a mixture of 5 plasmidswith the following molar ratios: 2× genomic plasmid (F1-0-03), 1×Rev-containing plasmid, 1× VSV-G-containing plasmid, 1×anti-CD3-scFvFc-GPI-containing plasmid, and 1× gag/pol-containingplasmid. To obtain lentiviral particles pseudotyped with VSV-G andexpressing anti-CD3-scFvFc-GPI and CD80-GPI on their surfaces, the totalDNA (1 μg/mL of culture volume) used was a mixture of 6 plasmids withthe following molar ratios: 2× genomic plasmid (F1-0-03), 1×Rev-containing plasmid, 1× VSV-G-containing plasmid, 1× anti-CD3-scFvFccontaining plasmid, 1× CD80-containing plasmid, and 1×gag/pol-containing plasmid. Separately, the PEI was diluted in 1.5 mlGibco™ Opti-MEM™ to 2 μg/mL (culture volume, 2:1 ratio to DNA). After a5-minute room temperature incubation, the two solutions were mixedtogether thoroughly, and incubated at room temperature for 20 moreminutes. The final volume (3 ml) was added to the cells. The cells werethen incubated at 37° C. for 72 hours with rotation at 125 rpm and with8% CO₂. The antiCD3-scFvFc-GPI containing plasmids included scFvsderived from either OKT3 or UCHT1, and a GPI anchor attachment sequence.The UCHT1scFvFc-GPI vector encodes a peptide (SEQ ID NO:278) thatincludes human Ig Kappa signal peptide (amino acids 1-22 of NCBI GI No.CAA45494.1) fused to the UCHT1 scFv (amino acids 21-264 of NCBI GI No.CAH69219.1), fused to human IgG1 Fc (amino acids 1-231 of NCBI GI No.AEV43323.1) with an A to T substitution at position 115, fused to thehuman DAF GPI anchor attachment sequence (amino acids 345-381 of NCBI GINo. NP_000565). The OKT3scFvFc-GPI vector encodes a peptide (SEQ IDNO:279) that includes a human Ig Kappa signal peptide (amino acids 1-22of NCBI GI No. CAA45494.1) fused to the OKT3 scFv (SEQ ID NO:285) fusedto human IgG1 Fc (amino acids 1-231 of NCBI GI No. AEV43323.1) fused tothe human DAF GPI anchor attachment sequence (amino acids 345-381 ofNCBI GI No. NP_000565). The CD80-GPI vector encodes a peptide (SEQ IDNO:280) that includes the human CD80 signal peptide and extracellulardomain (amino acids 1-242 of NCBI GI No. NP_005182) fused to the humanCD16b GPI anchor attachment sequence (amino acids 196-233 of NCBI GI No.NP_000561).

After 72 hours, the supernatants were harvested and clarified bycentrifugation at 1,200 g for 10 minutes. The clarified supernatantswere decanted to a new tube. The lentiviral particles were precipitatedby overnight centrifugation at 3,300 g, at 4° C. The supernatant wasdiscarded, and the lentiviral particle pellets were resuspended in 1:100of initial volume of X-Vivo™ 15 medium (Lonza). Lentiviral particleswere titered by serial dilution and analysis of GFP expression, in 293Tand Jurkat cells, 72 hours post-transduction, by flow cytometry.

Peripheral blood mononuclear cells (PBMCs) were first isolated fromeither fresh blood in ACD (acid citrate dextrose) tubes, for Donors 12Fand 12M, or from a buffy coat for Donor 13F, collected and distributedby the San Diego Blood Bank, CA. SepMate™ 50 (Stemcell™)_based gradientdensity separation of PBMCs on Ficoll-Paque PLUS® (GE Healthcare LifeSciences) was performed per manufacturers' instructions. 30 mL of bloodor buffy-coat diluted in PBS-2% HIFCS (heat inactivated fetal calfserum) were layered per each SepMate™ tube. After centrifugation at roomtemperature, at 1,200 g, for 20 min, the PBMC layers were collected,pooled and washed three times with 45 mL of PBS-2% HIFCS andcentrifugation at 400 g for 10 min at room temperature. The pellets werethen incubated at room temperature for 10 min in 10 mL of RBC lysisbuffer (Alfa Aesar) and washed an additional two times with 45 mL ofPBS-2% HIFCS, and centrifugation at 400 g for 10 min at roomtemperature. A final wash was performed in the transduction and culturemedia: X-Vivo™ 15 for Donor 13F, or RPMI-1640+10% HIFCS for Donors 12Fand 12M. No additional steps were taken to remove monocytes. Afterisolation, fresh and unstimulated PBMCs were resuspended to a finalconcentration of 1×10⁶/mL in their respective medium, and weretransduced, in duplicates or triplicates, with the lentiviral particlesdisclosed previously. The transductions were conducted for 14h, at 37°C., 5% CO₂, in X-Vivo™ 15 medium for Donor 13F, or in RPMI-1640+10%HIFCS for Donors 12F and 12M. Transductions were usually conducted atMOI 1 in a 12 wells plate format, 1 mL/well. For the kinetic experiment,0.5×10⁶ PBMCs/mL were transduced in 7 mL final, at MOI 1, in a 125 mLshake flask incubated at 37° C. for 2-20h hours with rotation at 125 rpmand with 8% CO₂. After incubation with the retroviral particles for theselected time, the cells were washed three times with X-Vivo™ 15 mediumfor Donor 13F, or PBS+2% HIFCS for Donors 12F and 12M, and finallyincubated at a cell density of 1×10⁶/mL in X-Vivo™ 15 medium for Donor13F, or RPMI-1640+10% HIFCS for Donors 12F and 12M, at 37° C., 5% CO₂.Starting at day 3 post transduction, and every 2-3 days from then on,the volume of the medium was doubled and supplemented with IL-2 to afinal concentration of 100 U/mL. Samples were collected at various dayspost-transduction (Day 3-17) to evaluate, by GFP expression levels, thetransduction efficiencies of each type of lentivirus that was generated.

At various days post-transduction, for lentiviral particles pseudotypedwith VSV-G, with or without OKT3 antibody (Biolegend) at 1 μg/ml;lentiviral particles pseudotyped with VSV-G and expressing anti-CD3scFvFc-GPI on their surface; or lentiviral particles pseudotyped withVSV-G and expressing anti-CD3 scFvFc-GPI and CD80-GPI on their surface;100 μL of cells were collected and analyzed by flow cytometry forexpression of GFP in the CD3+ cell population.

FIG. 21A and FIG. 21B show a histogram of the percentage (%) CD3+ GFP+cells in the total CD3+ population and a histogram of the absolute cellcount per well of the CD3+ GFP+ population, respectively, at 3, 6, 9, 13and 17 days after transduction of freshly isolated and unstimulatedPBMCs from Donor 12M, for 14h with the indicated lentiviral particles.Each bar represents the mean+/−SD of duplicates. FIGS. 21A and 21B showthat pseudotyping lentiviral particles with VSV-G and expressingantiCD3-scFvFc on the surface of the lentiviral particles effectivelytransduces freshly isolated and unstimulated PBMCs. Anti-CD3 scFv'sderived from either OKT3 or UCHT1, when in the form of an scFvFc-GPI,were effective.

FIG. 22A and FIG. 22B show a histogram of the percentage (%) CD3+ GFP+cells in the total CD3+ population and a histogram of the absolute cellcount per well of the CD3+ GFP+ population, respectively, at 3 and 6days after transduction of freshly isolated and unstimulated PBMCs fromDonor 13F, for 14h, with the indicated lentiviral particles. Please notethat “A” are results using VSV-G pseudotyped lentiviral particles(triplicate experiments); “B” are results using VSV-G pseudotypedlentiviral particles with OKT3 antibody (1 μg/mL) added to thetransduction medium (duplicate experiments); “C” are results using VSV-Gpseudotyped lentiviral particles expressing GPI-anchored UCHT1scFvFc ontheir surface (triplicate experiments); and “D” are results using VSV-Gpseudotyped lentiviral particles expressing GPI anchored UCHT1scFvFc andGPI-anchored CD80 on their surface (duplicate experiments). Each barrepresents the mean+/−SD of duplicates or triplicates, as indicated inFIG. 22A. FIGS. 22A and 22B show that pseudotyping lentiviral particleswith VSV-G and expressing antiCD3-scFvFc-GPI and CD80-GPI on theirsurfaces also effectively transduces freshly isolated and unstimulatedPBMCs when the transduction is performed for 14 hours.

FIGS. 23A and 23B show a histogram of percentage (%) CD3+ GFP+ cells inthe total CD3+ population and a histogram of the absolute cell count perwell of the CD3+ GFP+ population, respectively, at 3, 6 and 9 days aftertransduction of freshly isolated and unstimulated PBMCs from Donor 12Mfor the indicated time of exposure (2-20h) (i.e. contacting of cellswith lentiviral particles in transduction reaction mixture), with theindicated lentiviral particles. Transduction was performed in a plate ora shaker flask as indicated. Each bar represents the mean+/−SD ofduplicates for lentiviral particles pseudotyped with VSV-G (“[VSV-G]”);the other experiments did not have replicates. FIGS. 23A and 23B showthat freshly isolated and unstimulated PMBCs can be effectivelytransduced in as few as 2 hours with lentivirus particles pseudotypedwith VSV-G and expressing anti-CD3 scFvFc and CD80 on their surfaces.

It is noteworthy that subsequent experiments were performed usinglentiviral particles produced using only Rev, VSV-G, gag/pol, andF1-0-03 (and not a vector encoding an antiCD3 or CD80) to transducefreshly isolated resting PBMCs in the presence of soluble anti-CD3antibodies (100 ng/ml) for 2.5 hours. Transduction was achieved using 3different soluble anti-CD3 antibody clones. Transduction efficiencies asmeasured by CD3+ GFP+ cells at Day 6 were 4.31%, 3.27%, and 0.52% forCD3 antibody clones HIT3A, OKT3, and UCHT1, respectively. Backgroundtransduction efficiencies in the absence of soluble antibodies was0.06%.

Example 13. Functionality of miRNAs Inserted into the EF-1Alpha PromoterIntron

Four separate gBlocks® Gene Fragments were designed, each containing amiR-155 framework, including a miR-155 5′ flanking sequence or “5′ arm”(SEQ ID NO:256) and a miR-155 3′ flanking sequence or “3′ arm” (SEQ IDNO:260). For each gBlock®, a unique miRNA fragment targeting the CD3zetamRNA transcript was used to replace the miR-155 stem-loop precursor.Each gBlock® contained a 40 bp overlap sequence designed to facilitateassembly of all four gBlocks® as a single chain into the EF-1alphapromoter intron. The gBlocks® were assembled using a commercial kit forperforming Gibson® assembly ultra (NEBuilder, New England Biolabs,Inc.).

The synthetic EF-1alpha promoter and intron A containing the miRNAs (inSEQ ID NO:255) was part of a transgene expression cassette drivingexpression of GFP and eTag contained in a lentivirus vector backbone(the lentivirus vector backbone with the GFP and exemplary eTagrecognized by cetuximab is referred to herein as F1-0-02; FIGS. 24A and24B). The nucleotide positions of each gBlock® and its respectivecomponents in SEQ ID NO:255 are denoted in Table 3 as are the positionsof each “Feature” in FIG. 24B. Proper assembly of four miRNA into thelentivirus vector backbone was confirmed by comprehensive sequencing ofthe modified EF-1alpha promoter and intron region.

TABLE 3 Nucleotide positions of features in SEQ ID NO: 255 Nucleotidepositions Feature in SEQ in Feature ID NO: 255 SEQ ID NO: FIG. 24BgBlock® 1  927-1138 EF1alpha 927-966  1 overlap miR155- 967-994SEQ ID NO: 256  2 5′ arm CTGGAGGCTTGCTG AAGGCTGTATGCTG miRNA1-  995-1015SEQ ID NO: 257  3 5′ Stem ACATGGTACAGTTC AATGGTG miR loop 1016-1034SEQ ID NO: 258  4 GTTTTGGCCACTGA CTGAC miRNA1- 1035-1053 SEQ ID NO: 259 5 3′ Stem CACCATTGCTGTAC CATGT miR155- 1054-1098 SEQ ID NO: 260  63′ arm CAGGACACAAGGCC TGTTACTAGCACTC ACATGGAACAAATG GCC gBlock® 21099-1310 40 bp 50% 1099-1138  7 GC Linker 1 miR155- 1139-1166SEQ ID NO: 256  2 5′ arm miRNA2- 1167-1187 SEQ ID NO: 261  8 5′ StemTCAGTCTGTTCATC TTCTGGC miR loop 1188-1206 SEQ ID NO: 258  4 miRNA2-1207-1225 SEQ ID NO: 262  9 3′ Stem GCCAGAAGGAACAG ACTGA miR155-1226-1270 SEQ ID NO: 260  6 3′ arm gBlock® 3 1271-1482 40 bp 50%1271-1310  7 GC Linker 2 miR155- 1311-1338 SEQ ID NO: 256  2 5′ armmiRNA3- 1339-1359 SEQ ID NO: 263 10 5′ Stem AAGCGTGAAGTGAA TCAACGGmiR loop 1360-1378 SEQ ID NO: 258  4 miRNA3- 1379-1397 SEQ ID NO: 264 113′ Stem CCGTTGATACTTCA CGCTT miR155- 1398-1442 SEQ ID NO: 260  6 3′ armgBlock® 4 1443-1654 40 bp 50% 1443-1482  7 GC Linker 4 miR155- 1483-1510SEQ ID NO: 256  2 5′ arm miRNA4- 1511-1531 SEQ ID NO: 265 12 5′ StemGCAGTATCCTAGTA CATTGAC miR loop 1532-1550 SEQ ID NO: 258  4 miRNA4-1551-1569 SEQ ID NO: 266 13 3′ Stem GTCAATGTTAGGAT ACTGC miR155-1570-1614 SEQ ID NO: 260  6 3′ arm EF-1alpha 1615-1654 overlap

Replication incompetent lentiviral particles containing a nucleic acidencoding the four miRNAs directed against CD3zeta in their genome wereproduced by transient co-transfection of four plasmids into suspensionHEK293 cells: a plasmid containing the nucleic acid encoding F1-0-02modified to include the four miRNAs targeting the CD3zeta mRNAtranscript, a plasmid encoding VSV-G, a plasmid encoding REV, and aplasmid encoding GAG-POL. Lentiviral particle supernatant was harvestedafter 48 hours and PEG-precipitated for 24 hours. Supernatants werecentrifuged, and pelleted lentivirus particles were resuspended incomplete PBMC growth media without IL-2. Lentivirus particle titers werecalculated by 48 hour transduction of Jurkat cells.

For transduction, PBMCs were thawed on Day 0 and incubated for 24 hourswith 100U/mL of hrIL-2. On Day 1, PBMCs were activated via CD3/CD28conjugated beads. On Day 2, activated PBMCs were transduced with thelentiviral particles containing a genome with a nucleic acid sequenceencoding the miRNAs at an MOI of 10. Cells were expanded until Day 11,with fresh hrIL-2 added every two days. On days 7, 9, and 11, 1 millioncells were harvested for FACS analysis.

Cells were stained for CD3 Epsilon surface expression, using PEconjugated OKT-3 antibody (Biolegend). Expression levels were determinedby the mean fluorescence intensity (MF) of PE in the GFP positivepopulation (transduced cells). Expression levels of transduced cellswere compared between retroviral particles derived from F1-0-02 andretroviral particles derived from F1-0-02 in which the nucleic acidsequence encoding the CD3z miRNAs positioned in series were insertedinto the EF-1alpha promoter and intron A.

Results are shown in FIG. 25 . This data shows that serial miRNAstargeting CD3zeta encoded by a nucleic acid sequence within theEF-1alpha promoter intron A, are effective at knocking down expressionof the CD3 complex.

Example 14. Positional Independence of Serial Inhibitory RNAs Insertedinto the EF-1Alpha Promoter Intron Cloning

Four miRNA-expressing lentiviral vector constructs were designed to testthe processing of individual miRNA precursors in a structure comprising4 miRNA precursors in series. Table 4 shows the names of the individualconstructs and the position of the miR-TCRα in each construct.

TABLE 4 Constructs containing polycistronic miRNAs Construct Position 1Position 2 Position 3 Position 4 TCRa-P1 miR-TCRa miR-155 miR-PD-1miR-CTLA-4 TCRa-P2 miR-155 miR-TCRa miR-PD-1 miR-CTLA-4 TCRa-P3 miR-155miR-PD-1 miR-TCRa miR-CTLA-4 TCRa-P4 miR-155 miR-CTLA-4 miR-PD-1miR-TCRa

Each miRNA contained the miR-155 framework used in Example 13, i.e. amiR-155 5′ arm (SEQ ID NO:256), a miR-155 3′ arm (SEQ ID NO:260), a loop(SEQ ID NO:258), and a specific order of stem sequences as shown inTable 5. The type IIs assembly method was used to achieve assembly ofthe four miRNA fragments into their appropriate positions within theEF-1alpha intron of the lentivirus vector construct (F1-0-02; providedin Example 13 and shown in FIG. 24A).

TABLE 5 Sequences in miRNA constructs TCRa-P1 TCRa-P2 TCRa-P3 TCRa-P4(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 278) NO: 279) NO: 280) NO: 281)5′ arm SEQ ID SEQ ID SEQ ID SEQ ID NO: 256 NO: 256 NO: 256 NO: 256miRNA1-5′ SEQ ID SEQ ID SEQ ID SEQ ID Stem NO: 267 NO: 270 NO: 270NO: 270 miR loop SEQ ID SEQ ID SEQ ID SEQ ID NO: 258 NO: 258 NO: 258NO: 258 miRNA1-3′ SEQ ID SEQ ID SEQ ID SEQ ID Stem NO: 268 NO: 271NO: 271 NO: 271 3′ arm SEQ ID SEQ ID SEQ ID SEQ ID NO: 260 NO: 260NO: 260 NO: 260 Linker 1 SEQ ID SEQ ID SEQ ID SEQ ID NO: 269 NO: 269NO: 269 NO: 269 5′ arm SEQ ID SEQ ID SEQ ID SEQ ID NO: 256 NO: 256NO: 256 NO: 256 miRNA2-5′ SEQ ID SEQ ID SEQ ID SEQ ID Stem NO: 270NO: 267 NO: 273 NO: 276 miR loop SEQ ID SEQ ID SEQ ID SEQ ID NO: 258NO: 258 NO: 258 NO: 258 miRNA2-3′ SEQ ID SEQ ID SEQ ID SEQ ID StemNO: 271 NO: 268 NO: 274 NO: 277 3′ arm SEQ ID SEQ ID SEQ ID SEQ IDNO: 260 NO: 260 NO: 260 NO: 260 Linker 2 SEQ ID SEQ ID SEQ ID SEQ IDNO: 272 NO: 272 NO: 272 NO: 272 5′ arm SEQ ID SEQ ID SEQ ID SEQ IDNO: 256 NO: 256 NO: 256 NO: 256 miRNA3-5′ SEQ ID SEQ ID SEQ ID SEQ IDStem NO: 273 NO: 273 NO: 267 NO: 273 miR loop SEQ ID SEQ ID SEQ IDSEQ ID NO: 258 NO: 258 NO: 258 NO: 258 miRNA3-3′ SEQ ID SEQ ID SEQ IDSEQ ID Stem NO: 274 NO: 274 NO: 268 NO: 274 3′ arm SEQ ID SEQ ID SEQ IDSEQ ID NO: 260 NO: 260 NO: 260 NO: 260 Linker 3 SEQ ID SEQ ID SEQ IDSEQ ID NO: 275 NO: 275 NO: 275 NO: 275 5′ arm SEQ ID SEQ ID SEQ IDSEQ ID NO: 256 NO: 256 NO: 256 NO: 256 miRNA4-5′ SEQ ID SEQ ID SEQ IDSEQ ID Stem NO: 276 NO: 276 NO: 276 NO: 267 miR loop SEQ ID SEQ IDSEQ ID SEQ ID NO: 258 NO: 258 NO: 258 NO: 258 miRNA4-3′ SEQ ID SEQ IDSEQ ID SEQ ID Stem NO: 277 NO: 277 NO: 277 NO: 268 3′ arm SEQ ID SEQ IDSEQ ID SEQ ID NO: 260 NO: 260 NO: 260 NO: 260 where: SEQ ID NO: 267= TCRalpha miRNA Stem 1; ATATGTACTTGGCTGGACAGC SEQ ID NO: 268 = TCRalphamiRNA Stem 2; GCTGTCCACAAGTACATAT SEQ ID NO: 269 = Linker1;CACATTGGTGCCGGATGAAGCTCTTATGTTGCCGGTCAT SEQ ID NO: 270 = mir-155 Stem 1;CTGTTAATGCTAATCGTGATA SEQ ID NO: 271 = mir-155 Stem 2;TATCACGATTATTAACAG SEQ ID NO: 272 = Linker2;GTTGCCGGAGTCTTGGCAGCGAGAGATCACTATCAACTAA SEQ ID NO: 273 = PD-1 miRNAStem 1; TACCAGTTTAGCACGAAGCTC SEQ ID NO: 274 = PD-1 miRNA Stem 2;GAGCTTCGCTAAACTGGTA SEQ ID NO: 275 = Linker3;GTGTTAATTGTCCATGTAGCGAGGCATCCTTATGGCGTGG SEQ ID NO: 276 = CTLA-4 miRNAStem 1; TGCCGCTGAAATCCAAGGCAA SEQ ID NO: 277 = CTLA-4 miRNA Stem 2;TTGCCTTGTTTCAGCGGCA

Lentiviral Particle Production

The four constructs and the control F1-0-02, which includes no nucleicacid sequence encoding a miRNA, were used to produce lentiviralparticles in 30 mL suspension cultures of 293T cells. The lentiviralparticles were harvested and concentrated by PEG precipitation.Functional lentiviral particle titers were obtained by transducingJurkat cells at multiple dilutions (1:1000, 1:10000, 1:100000),incubating the lentiviral particles and cells for 2 days at 37° C.,washing the cells 2× with FACS buffer, and analyzing for GFP by flowcytometry. Other details regarding lentiviral particle production areprovided in Example 13 herein.

Transduction

For transduction, PBMCs were thawed and recovered overnight in completemedia containing 100U/mL hrIL-2. 1×10⁵ PBMCs were activated via exposureto CD3/CD28 conjugated beads for 24 hours. Cells were transduced induplicate wells with each of the four miRNA constructs or with controlretroviral particle F1-0-02 at MOI 10. The cells were supplied with100U/mL hrIL-2 every 3 days and expanded until day 10.

FACS

Cells were harvested for FACS analysis which confirmed that the cellswere transduced with the replication incompetent lentiviral vectors. Theresults showed that approximately equivalent amounts of miRNA containingvirus were delivered to each well in the experiment.

Cells-to-Ct miRNA RT-qPCR and Analysis

An RT-qPCR assay was designed to detect expression and processing of themiRNA precursors into mature processed miRs. Analysis was done by firstnormalizing all miR-TCRα ct values to the RNU48 internal control toproduce ΔCt values. Next, the ΔCt values of each transduced sample weresubtracted from the ΔCt of the non-transduced control to produce ΔΔCt.This value is representative of the amount of processed miR-TCRα miRNAin each transduced sample, relative to the non-transduced control.

As shown in FIG. 26 , the RT-qPCR assay successfully detected processedmiR-TCRα in samples transduced with miR-TCRα containing replicationincompetent lentiviral particles. Furthermore, the results clearlyindicate that there is no remarkable difference in miRNA TCRα processingat any of the four positions tested.

Example 15. Cytotoxic Activity of Microenvironment Restricted BiologicCAR-Expressing T Cells can be Controlled by Changing pH

The following example illustrates how the cytotoxic activity oftransduced T cells (also referred to as effector cells) expressingMRB-CARs can be modulated by changes in the pH of the microenvironment.In this example, nucleic acids encoding an MRB-CAR capable of bindingthe cognate antigen Target 1 (anti-Target 1) were used to generatereplication incompetent recombinant lentiviral particles. Pan T cellswere transduced with the lentiviral particles and the cytotoxic activityof the effector cells were compared using Real-Time Cell Analysis (RTCA)before and after changing the pH of the media.

Production of Replication Incompetent Recombinant Lentiviral Particles

A nucleic acid that encoded T1B, an anti-Target 1 MRB-CAR from theseries of candidate MRB-CARs in Example 3, was tested. Replicationincompetent recombinant lentiviral particles were produced by transienttransfection of Lenti-X 293T cells (Clontech, Mountain View, CA) withlentiviral expression vectors and nucleic acids that included segmentsencoding either the MRB-CAR or a control, Cl, that contained a GMCFspand an eTAG (SEQ ID NO:284), but did not include an anti-Target 1MRB-CAR. The cells were adapted to suspension culture by serial growthin Freestyle 293 Expression Medium (ThermoFisher Scientific, Waltham,MA). The cells in suspension were transfected using PEI (Polysciences,Warminster, PA) dissolved in weak acid. Cells (30 mL) were grown to1×10⁶ cells/mL in a 125 mL Erlenmeyer flask.

Total DNA was diluted in 1.5 ml Optimem media for 30 mL of cells. TotalDNA (1 μg/mL of culture volume) was a mixture of 4 plasmids with thefollowing molar ratios: 2× genomic plasmid that included lentiviralpackaging elements, LTRs and the nucleic acid encoding T1B, 1×Rev-encoding plasmid, 1× VSVg-encoding plasmid, and 1× Gagpol-encodingplasmid. Separately, the PEI was diluted in 1.5 ml Optimem to 2 μg/mL(culture volume, 2:1 ratio to DNA). After a 5 minute room temperatureincubation, the two solutions were mixed together well and incubated atroom temperature for 20 minutes. The final volume (3 ml) was added tothe cells. The cells were then incubated at 37° C. for 72 hours withrotation at 120 rpm and with 5-8% CO2.

After 72 hours, the supernatant was harvested by centrifugation at 1,000g for 10 minutes. The supernatant was decanted to a fresh tube and ¼ ofthe supernatant volume in PEG solution (PEG-IT, System Biosciences) wasadded. The replication incompetent recombinant lentiviral particles wereprecipitated by incubation overnight at 4° C. followed by centrifugationat 1,500 g for 20 minutes at 4° C. The supernatant was removed, and thevirus was resuspended in 1:100 volume of X-VIVO 15 media. Viruses weretitered by eTAG expression in Jurkat cells.

T Cell Transduction/Expansion

Pan T cells were obtained from AllCells. Anti-Target 1 MRB-CARreplication incompetent recombinant lentiviral particles were made asdiscussed above. Two days prior to lentiviral transduction, cells werethawed and cultured in X-VIVO 15 media (Lonza, Basel, Switzerland) with5% human AB serum (Valley Biomedical Inc., Winchester, VA) and 10 mMN-acetyl L-Cysteine (Sigma-Aldrich, St. Louis, MO). Recombinant humanIL-2 (R&D Systems, Minneapolis, MN) was added to a final concentrationof 100 IU/mL. Twenty-four hours prior to viral transduction, primaryhuman T cells were seeded into a 12-well plate at 0.5×10⁶ cells/well andactivated using Dynabeads Human T-Activator CD3/CD28 (ThermoFisherScientific) at a 1:3 cell:bead ratio. On the day of transduction, thelentiviral particle solution was added to the wells at an MOI of 5.Transduced Pan T cells were maintained at ˜10⁶/mL in X-VIVO 15 media for3 days, then transferred into a 6-well G-Rex plate with 30 mL/well ofX-VIVO 15 media with 100 IU/mL IL-2. Cells were cultured for at least 10days before experiments were conducted and IL-2 was added every otherday.

pH Shift Cytotoxicity Assay

The cytotoxic activity of transduced T cells before and after pH changeby addition of NaHCO₃ or NaOH was measured using the xCELLigence System.Briefly, one day before the experiment, target cells (CHO cells stablytransfected with a construct to express Target 1 on the cell surface(CHO-Target 1 cells)), were seeded into a 96-well E-plate (ACEA; SanDiego, CA) at 10,000 cells/well with X-VIVO 15 media containing 40 mMHEPES and 40 mM PIPES, pH 6.7. Cryopreserved effector cells previouslytransduced with either lentiviral particles containing the nucleic acidencoding T1B or Cl (T1BVP and C1VP, respectively) produced as discussedabove, were thawed and cultured for two days in X-VIVO 15 mediacontaining 100 IU/mL of IL-2 (R&D Systems, Minneapolis, MN). On the dayof the experiment, cells transduced with T1BVP or C1VP were washed andresuspended in X-VIVO 15 media containing 40 mM HEPES and 40 mM PIPES,pH 6.7 and then added into the experimental wells at effectorcell/target cell ratios (E/T) of 1:1.

Impedance readings measured on the xCELLigence System (ACEA) were takenevery 5 minutes and reported as the Cell Index (CI) to quantitate cellconfluency as a measure of cell proliferation/cell lysis. Approximately3 hours after effector cell addition, 8 μl of 7.5% NaHCO₃ or 14 μl of0.5 M NaOH was added into the wells with X-VIVO 15 media containing 40mM HEPES and 40 mM PIPES, pH 6.7 to increase the pH from 6.7 to 7.4.Impedance readings were continued for approximately 20 hours aftereffector cell addition. Percentage of specific cytolysis was calculatedas follows ((CI Target+C1VP transduced effector T cells)−(CITarget+T1BVP transduced effector T cells))/(CI Target+C1VP transducedeffector T cells)×100.

HCl Switch on RTCA Killing Assay

The cytotoxic activity of transduced T cells before and after pH changeby addition of HCl was measured using the xCELLigence System. Briefly,one day before the experiment, CHO-Target 1 cells were seeded into a 96well E-plate at 10,000 cells/well with X-VIVO 15 media containing 40 mMHEPES and 40 mM PIPES, pH 7.4. Cryopreserved effector cells previouslytransduced with either C1VP or T1BVP, were thawed and cultured for twodays in X-VIVO 15 media containing 100 IU/mL of IL-2. On the day of theexperiment, cells transduced with T1BVP or C1VP were washed andresuspended in X-VIVO 15 media containing 40 mM HEPES and 40 mM PIPES,pH 7.4 and then added into experimental wells at effector cell/targetcell ratios (E/T) of 1:1.

Impedance readings were taken every 5 minutes and reported as the CellIndex (CI). Approximately 3 hours after effector cell addition, 8 μl of1 M HCl was added into the wells with X-VIVO 15 media containing 40 mMHEPES and 40 mM PIPES, pH 7.4 to switch the pH from 7.4 to 6.7.Impedance readings were continued for approximately 20 hours aftereffector cell addition. Percentage of specific cytolysis was calculatedas follows ((CI Target+C1VP transduced effector T cells)−(CITarget+T1BVP transduced effector T cells))/(CI Target C1VP)×100.

Results

The cytotoxic activity of an MRB-CAR capable of binding cognate antigenTarget 1 with increased activity at a reduced pH was compared in pH 6.7and pH 7.4. T cells that were transduced with lentiviral particlesencoding an anti-Target 1 MRB-CAR were used to kill CHO cells expressingTarget 1, and then the pH was increased to determine whether thecytotoxic activity could be inhibited by a pH shift. As shown in FIGS.27A and 27B, the addition of either NaHCO₃ or NaOH to themicroenvironment of active CAR-T cells to increase the pH of the mediainhibited the cytotoxic activity of the T cells expressing the MRB-CAR.These results show that active MRB-CAR expressing T cells can killtarget-expressing cells and then this killing activity can be inhibitedby increasing the pH of the microenvironment.

The ability of the cytotoxic activity of T cells expressing the MRB-CARto be activated by a pH change was also determined. As shown in FIG.27C, the cytotoxic activity of anti-Target 1 MRB-CAR expressing T cellson CHO-Target 1 cells was low at a pH of 7.4 and was increased by theaddition of HCl to reduce the pH of the microenvironment. Cumulatively,these results demonstrate the cytotoxic activity of T cells expressingMRB-CARs can be modulated by a shift in pH within the microenvironment,both by reducing cytotoxic activity after an increase in pH andincreasing cytotoxic activity after a decrease in pH. In thisnon-limiting example, pH was increased from pH 6.7 and decreased from7.4.

Example 16. Bicarbonate Administration can Increase pH of the TumorMicroenvironment in Mice

The following example demonstrates the pH of an in vivo tumormicroenvironment can be modulated by administering a pharmacologicagent. In this example, the pharmacologic agent is sodium bicarbonateand the tumor microenvironment is a CHO xenograft tumor in mice. Theexample includes two methods of measuring the pH of a tumormicroenvironment, both in vivo and ex vivo.

The extracellular microenvironment of most solid tumors is acidic, witha pH typically between 6.5 and 6.9. On the contrary, normal tissue pH isbasic, with a pH typically between 7.2 and 7.5. However, directlymeasuring the in vivo pH of a tumor microenvironment can be difficult.Fortunately, the relative protease activity of cathepsin is higher atlower pH and lower at higher pH. Therefore, the measurement ofintratumoral cathepsin activity can serve as a surrogate measure of thepH of the tumor microenviroment. To measure in vivo activities ofcathepsin B, L, S, K, V, and D, the near-infrared ProSense 750 FASTprobe (PerkinElmer) was used. To further confirm modulation of the pH inthe tumor microenvironment by administration of sodium bicarbonate,excised tumors were treated with phenol red and the color was noted.Phenol red is a pH indicator which undergoes a pH-dependent colortransition. The sodium salt of phenol red is widely used in cell culturemedia to identify pH values. A solution of phenol red has a yellow colorat a pH of 6.4 or below, an orange color around pH 7.0, a red coloraround pH 7.4, and a purple color above pH 7.8.

Mice were handled in accordance with Institutional Animal Care and UseCommittee approved protocols. Subcutaneous (sc) Chinese Hamster Ovary(CHO) tumor xenografts were established in the hind flank of 12-14 weekold female B-NSG mice (NOD-PrkdcscidIl2rgtml/Bcgen (Beijing BiocytogenCo. Ltd.). Briefly, cultured CHO cells (ATCC, Manassas, VA) were washedin DPBS (ThermoFisher), counted, resuspended in cold DPBS and mixed withan appropriate volume of Matrigel ECM (Corning; final concentration 5mg/mL) at a concentration of 1.5×10⁶ cells/200 μl on ice.

Animals were prepared for injection using standard approved anesthesiawith hair removal (Nair) prior to injection. 200 μl of the cellsuspension in ECM was injected sc into the rear flanks of the mice. Oncetumors were palpable, the tumors were measured using calipers 2times/week. Tumor volume was calculated using the following equation:(longest diameter*shortest diameter²)/2. When average tumor volumereached 200 mm³, mice were randomly assigned to the respective treatmentgroups.

Two days before the administration of bicarbonate, the drinking waterfor the B-NSG mice was changed from acidic to regular pH autoclavedpurified water. The following day, the 750 ProSense FAST probe wasadministered to 6 CHO-xenograft tumor bearing mice via 100 μl tail veininjections (4 nmol ProSense 750 FAST probe/100 μlμl PBS). A separategroup of CHO-xenograft tumor bearing mice was left untreated. Thefollowing day, sodium bicarbonate was administered and imaging of themice treated with the ProSense 750 FAST probe was performed using aCaliper IVIS Lumina XR. Briefly, mice were anesthetized using 3% O₂ 2L/min isoflurane in 02 carrier gas at 2 L/min and then placed with nosecones supplying 1.5% isoflurane to anesthetized mice during imaging.Image acquisitions consisted of a 5 sec exposure for near-infraredprobes (745/810 nm excitation/emission wavelength). Fluorescence imageswere overlaid on normal light images of the mice. Time 0 (pretreatment)images were acquired before administration of either PBS (control) orsodium bicarbonate. The mice were then administered either 1 ml/mousePBS (control, ThermoFisher) or 1 ml/mouse 1 M sodium bicarbonate(Shanghai Experiment Reagent Co., LTD) via intraperitoneal injection(ip). Mice were then imaged at 30 min post administration of PBS orbicarbonate. The collected fluorescence images were adjusted to haveidentical minimums, maximums, and threshold values. The photon countswere defined in this study as relative fluorescence units (RFU). RFU wascalculated by normalizing the photon counts from the 30 min time pointto the pretreatment time point (time 0; 100%) in each mouse. Due tovariability between fluorescence values in each mouse at the time 0pretreatment value, the observed fluorescence intensity values atdifferent time points were normalized only to the individual mouse andnot to a mean pretreatment value.

In a separate arm of the experiment, the 6 mice that did not receive theNIR cathepsin probe were euthanized by cervical dislocation at 1.5 hourspost ip administration of PBS or sodium bicarbonate. The CHO xenografttumor was excised from each mouse. The xenograft tumors were split intotwo halves with a scalpel and placed on a petri dish. The tumor tissuehalves were then cut/sliced repeatedly using the scalpel. Water or 0.05%phenol red solution (50 mg phenol red/100 ml water) was added dropwiseto each tumor half, respectively. The color was noted and images weretaken of the treated tumor xenografts and of the phenol red solutionremaining on the petri plate once the tumor xenograft samples wereremoved.

Results

FIG. 29 shows the RFU results (mean with SEM) from imaging intratumoralcathepsin activity in CHO-xenograft tumor bearing mice before and afteradministration of PBS (control; n=3) or bicarbonate (n=3). These resultssuggest that sodium bicarbonate administration can increase the pH ofthe tumor microenvironment in vivo as evidenced by the decreasedcathepsin activity observed following ip sodium bicarbonateadministration.

A color change of the phenol red indicator from yellow/orange to red wasobserved using the tumor tissue excised from sodium bicarbonate-treatedmice (n=3) relative to the PBS-treated mice (n=3). These results suggestthat sodium bicarbonate administration increased the pH of the tumormicroenvironment in vivo following ip administration as evidenced by thecolor change of the phenol red indicator from yellow/orange to red.

Example 17. Identification of Candidate Chimeric PolypeptideLymphoproliferative Elements

In this example, two chimeric polypeptide libraries (Library 1 andLibrary 2) of candidate (putative) chimeric lymphoproliferative elementswere assembled into viral vectors from pools ofextracellular-transmembrane block sequences, intracellular blocksequences, and a barcode library according to the chimericpolypeptide-encoding constructs provided in FIGS. 30 and 31 . Theproliferation of library-transduced PBMCs was followed over time:genomic DNA was extracted from PBMCs at one-week intervals and thebarcode region amplified for DNA sequencing to estimate the number ofcells in these mixed cultures of PBMCs containing each of the testcandidate chimeric lymphoproliferative elements. Thus, the screen testedthe ability of the candidate chimeric lymphoproliferative elements topromote PBMC cell proliferation in the absence of IL-2 or any otherexogenous cytokine.

Two libraries were made and analyzed in this study: the constructs ofLibrary 1 (which was used in the screens identified as Libraries 1A,1.1A, and 1.1B) was flanked with a CAR at the 5′ end of the candidatechimeric polypeptide, while the constructs of Library 2 (which was usedin the screens identified as Libraries 2B and 2.1B) did not include aCAR (FIGS. 30 and 31 ). FIG. 30 provides a schematic of a non-limiting,exemplary transgene expression cassette containing a polynucleotidesequence encoding a CAR and a candidate chimeric lymphoproliferativeelement (CLE) of Library 1 driven by an EF-1 alpha promoter and aKozak-type sequence (GCCGCCACC (SEQ ID NO:519)), in a lentiviral vectorbackbone. The CAR was FLAG-tagged and contained an ASTR directed toCD19, a stalk and transmembrane portion of CD8, and an intracellularactivating domain from CD3z. Each candidate lymphoproliferative elementof Library 1 included 3 modules; an extracellular/transmembrane module(P1-2) and 2 intracellular modules (P3 and P4). The P1-2 module alsoencoded a recognition and/or elimination domain (TAG). A triple stopsequence (TAATAGTGA (SEQ ID NO:520)) separated P4 from a DNA barcode(P5). A WPRE(GTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGJGCTCTGCGGCCTCTTCCCCCTCTTCGCCTTCGCCCTCAGACCACTCGGATCTCCCTTTGGGCCCCCTCCCCGCC TG (SEQID NO:521)) was present between the last stop codon (starting 4 bp afterthe last nucleotide of the last stop codon) and the 3′ LTR (whichstarted 83 nucleotides after the last nucleotide of the WPRE).

The CAR and P1-2 of Library 1 were separated by a polynucleotidesequence encoding a T2A ribosomal skip sequence.

FIG. 31 provides a schematic of a non-limiting, exemplary transgeneexpression cassette containing a polynucleotide sequence encoding acandidate CLE of Library 2 driven by an EF-1 alpha promoter and aKozak-type sequence (GCCGCCACC(SEQ ID NO:519)), in a lentiviral vectorbackbone. Each candidate lymphoproliferative element of Library 2included 3 modules; an extracellular/transmembrane module (P1-2), and 2intracellular modules (P3 and P4). The P1-2 module also encoded arecognition and/or elimination domain (TAG). A triple stop sequence(TAATAGTGA(SEQ ID NO:520)) separated P4 from a DNA barcode (P5). A WPRE(GTCCTTTCCATGGCTGCTCGCCTGTCTTGCCACCTGGATTCTGCGCGGACGTCCTTCTGCTACCTCCCTTCGGCCCTCAATCCAGCGCACCTTCCTTCCCCCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCC TG (SEQID NO:521)) was present between the last stop codon (starting 4 bp afterthe last nucleotide of the last stop codon) and the 3′ LTR (whichstarted 83 nucleotides after the last nucleotide of the WPRE).

The assembled candidate chimeric polypeptide part of both librariesfollowed the exact same design with 3 positions (1extracellular/transmembrane module (P1-2) and 2 intracellular modules(P3 and P4), one of which could be an early termination signal). GGSlinkers encoded by nucleic acid sequences between each of the domainswere included as a product of construct assembly. Nucleic acids encodingfull or variant versions of either a Myc or an eTAG recognition and/orelimination domain (referred to in Table 6 as “eTAG” or “Myc Tag”) wereinserted 5′ to the extracellular domain of each truncated cytokinereceptor (or select LMP1 constructs) as part of the P1-2 modules, suchthat the encoded candidate chimeric polypeptides included a recognitionand/or elimination domain in frame with the extracellular domain. Asignal sequence was inserted at the 5′ terminus of the CAR-encodingsequences in Library 1 and at the 5′ terminus of the recognition and/orelimination domain-encoding sequences of Library 2. A Kozak-typesequence (GCCGCCACC (SEQ ID NO:519)), which is also as an enhancedKozak-type sequence, was inserted within 10 nucleotides before the ATGstart codon, which was at the 5′ terminus of the signal-sequenceencoding sequences in Library 1 and at the 5′ terminus the recognitionand/or elimination domain-encoding sequences (or select LMP1 constructs)in Library 2. A 4th module contained a random barcode generated byrandom PCR assembly of ˜10,000× ˜10,000 sub-barcodes (˜100 millioncombinations). The coding sequences of the CARs for Library 1 constructsencoded a signal sequence, a FLAG tag, an anti-CD19 ASTR scFv, a CD8astalk, a CD8a transmembrane domain, and a CD3zeta intracellularactivating domain. Expression of the CAR (Library 1 only) and CLEs(Library 1 and Library 2) was driven by an EF1 alpha promoter. In theconstructs of Library 1, a T2A site was positioned between the CAR andthe chimeric polypeptide encoding sequences. In libraries 1 and 2, atriple stop codon was present at the 3′ end of P4.

Synthesis of Viral Vectors

Individual parts for the library assembly were designed and synthesizedas DNA blocks with compatible Type IIs enzyme (AarI) overhangs andconnectors encoding a Glycine-Glycine-Serine sequence on the 5′ and 3′ends of each part. These parts were then individually cloned intouniversal plasmid vectors. The assembly for each part was done as a onetube assembly in which 0.04 μmol of the part, 0.04 μmol barcode mix, and0.04 μmol plasmid vector backbone were added into a 10 ul reactionfollowed by 1 unit AarI enzyme, 10 units T4 DNA ligase, and 1× ligasebuffer (50 mM Tris-HCl, 10 mM MgCl₂, 1 mM ATP, 10 mM DTT) for optimalligase activity. Digestion and ligation cycles were performed using athermocycler as follows: 55 cycles of 37° C. for 2 mins and 16° C. for 5mins; a 5 min incubation at 50° C. for final digestion; a 10 minincubation at 80° C. for AarI inactivation; and a final hold at 4° C.Then the assembled vectors were purified by conventional ethanolprecipitation. The resulting purified vectors were electroporated(Electro Micropulser, Bio-rad) into Top10 electro competent cells(Thermo Fisher Scientific) and directly recovered in liquid culturescontaining Kanamycin antibiotic for selection and expansion. Cultureswere incubated at 37° C. for 10-12 hours and then harvested and plasmidspurified using ZymoPURE-EndoZero™ Plasmid Gigaprep Kit.

Lentiviral Particle Production

Recombinant lentiviral particles were produced by transient transfectionof 293T cells (Lenti-X™ 293T, Clontech) with separate lentiviralpackaging plasmids encoding gag/pol and rev, and a pseudotyping plasmidencoding VSV-G. The synthesized viral vectors encoding candidatechimeric polypeptides with or without a co-expressed chimeric antigenreceptor were co-transfected with the packaging plasmids. The cells wereadapted to suspension culture by serial growth in Freestyle™ 293Expression Medium (ThermoFisher Scientific). The cells in suspensionwere seeded at 1×10⁶ cells/mL (200 mL) in a 1 L Erlenmeyer flask andimmediately transfected using polyethylenimine (PEI) (Polysciences)dissolved in weak acid.

Plasmid DNA was diluted in 10 ml Gibco™ Opti-MEM™ media for 200 mL ofcells. To obtain lentiviral particles pseudotyped with VSV-G, the totalDNA (1 μg/mL of culture volume) used was a mixture of 4 plasmids withthe following molar ratios: 2× combined viral vectors, 1× Rev-containingplasmid, 1× VSV-G-containing plasmid, and 1× gag/pol-containing plasmid.Separately, the PEI was diluted in 10 ml Gibco™ Opti-MEM™ to 2 μg/mL(culture volume, 2:1 ratio to DNA). After a 5-minute room temperatureincubation, the two solutions were mixed together thoroughly andincubated at room temperature for 20 minutes. The final volume (20 ml)was added to the cells. The cells were then incubated at 37° C. for 48hours with rotation at 125 rpm and with 8% CO₂.

After 48 hours, the supernatants were harvested and clarified bycentrifugation at 1,000 g for 10 minutes. Viral supernatant was thenfiltered through low protein-binding Millex-HV 0.45 m PVDF filters(Millipore, Catalog no. SLHVR25LS). The clarified supernatants weredecanted to a new tube, 1 volume of Lenti-X PEG (Takara, 4×) was mixedwith 3 volumes of clarified supernatant, and incubated at 4° C.overnight. The lentiviral particles were then precipitated by 90 mincentrifugation at 1,500 g and 4° C. The supernatant was discarded, andthe lentiviral particle pellets were resuspended in 1:100 of initialvolume of complete PBMC growth media without IL-2. Lentiviral particleswere titered using a p24 assay ELISA kit from Takara (#632200).

Transduction and Culturing of PBMCs

Whole human blood from a healthy donor (101 ml) was collected into a 200ml blood bag containing CDP anticoagulant (Nanger; 5-200). The blood wasprocessed using density gradient centrifugation with Ficoll-Pacque™(General Electric) using a Sepax 2 S-100 device (Biosafe; 14000)following manufacturer's instruction and kit CS900.2 (BioSafe; 1008), toobtain peripheral blood mononuclear cells (PBMCs). Yield of viable PBMCwas 7.3×10⁷ cells. 1.5×10⁷ PBMC were added each to two G-Rex 100M cellculture devices (Wilson Wolf, 81100S) to a final concentration of0.5×10⁶ viable cells/ml in a final volume of 30 ml of Complete OpTmizer™CTS™ T-Cell Expansion SFM (OpTmizer™ CTS™ T-Cell Expansion Basal Medium1 L (Thermo Fisher, A10221) supplemented with ml OpTmizer™ CTS™ T-CellExpansion Supplement (Thermo Fisher, A10484-02), CTS™ Immune Cell SR(Thermo Fisher, A2596101), and CTS™ GlutaMAX™-I Supplement (ThermoFisher, A1286001) according to manufacturer's instructions). Recombinanthuman interleukin-2 (IL-2) (Novoprotein) was also added to aconcentration of 100 IU/ml along with activating anti-CD3 Ab (OKT3,Novoprotein) at a concentration of 50 ng/ml, to activate the PBMCs forviral transduction. The G-Rex devices were incubated overnight in astandard humidified tissue culture incubator at 37° C. and 5% CO₂. Afterovernight incubation, lentivirus particle preparations containing thedesired test chimeric polypeptide-encoding constructs (Library 1 orLibrary 2) were added to individual G-Rex devices at a multiplicity ofinfection (MOI) of 5. The G-Rex devices (Library 1 and Library 2) wereincubated overnight in a standard humidified tissue culture incubator at37° C. and 5% CO₂. Following the overnight incubation, additionalComplete OpTmizer™ CTS™ T-Cell Expansion SFM was added to bring thefinal volume of each G-Rex device to 500 ml. No additional IL-2 or otherexogenous cytokine was added at this or following cell culture steps.The G-Rex® device was incubated in a standard humidified tissue cultureincubator at 37° C. and 5% CO₂ for 7 days from PBMC isolation. On day 7,the cells from the two G-Rex devices transduced with Library 1 orLibrary 2 were collected by centrifugation and resuspended into freshComplete OpTmizer™ CTS™ T-Cell Expansion SFM without IL-2. 1.25×10⁷ ofLibrary 1 cells and 2.54 10⁷ cells of Library 2 were placed in a newG-Rex 100M devices containing 500 ml Complete OpTmizer™ CTS™ T-CellExpansion SFM without IL-2. One vial of thawed Day 0 frozen donormatched PBMCs (1.46×10⁷ PBMC) was added to the G-Rex devices containingcells transduced with Library 1 to provide CD19+ B-cell activation ofthe CD19 ASTR. In these screens, cells fed with PBMCs were designatedwith an A after the library number, for example screens performed withcells transduced with Library 1 and fed with PBMCs are referred to asLibrary 1A herein. Library 2 received no Day 0 PBMCs. In these screens,cells not fed with PBMCs were designated with a B after the librarynumber, for example screens performed with cells transduced with Library2 and not fed with PBMCs are referred to as Library 2B herein. The G-Rexdevices were incubated overnight in a standard humidified tissue cultureincubator at 37° C. and 5% CO₂. On day 14, the cells from the two G-Rexdevices (Library 1A and Library 2B) were collected by centrifugation andresuspended into fresh Complete OpTmizer™ CTS™ T-Cell Expansion SFMwithout IL-2. These were each placed into individual wells of 6-wellG-rex plates (Wilson-Wolf; 80240M) containing 30 ml Complete OpTmizer™CTS™ T-Cell Expansion SFM without IL-2. One vial of thawed Day 0 frozendonor matched PBMCs was added to the well containing Library 1A cells toprovide CD19+ B-cell activation of the CD19 ASTR. The G-Rex devices wereincubated overnight in a standard humidified tissue culture incubator at37° C. and 5% CO₂. On day 21, the cells were collected from the wells ofthe 6-well G-rex plates, and the process performed on day 14 wasrepeated. Samples (1×10⁵ to ˜4-5×10⁶ cells) were collected at variousdays post-transduction (Days 7, 21, 28, and/or 35) and genomic DNA waspurified using GenElute™ Mammalian Genomic DNA Miniprep following thekits protocol. In some cases, samples were purified using densitygradient centrifugation with Ficoll-Pacque™ (General Electric) to removedead cells prior to genomic DNA extraction. Purified genomic DNA wassequenced using an Illumina HiSeq, generating paired-end 150 bp reads.Usually, a subset of 10 million reads was extracted from each indexedfastq file and processed for analysis using barcode_reader, a custom Rscript engineered to extract barcode sequences based on the presence ofa constant region. Purified genomic DNA was also sequenced on a PacBiosequencing system to obtain longer read lengths to associate barcodeswith constructs. These screens were repeated and the Libraries weredesignated 1.1A, 1.1B, and 2.1B, where the A and B indicated whether ornot the cells were fed with PBMCs as discussed above.

Data Analysis

Once HiSeq data was obtained for all time points, barcode count tableswere merged based on barcode ID. Counts were normalized to ‘Count permillion’ using the formula: 1×10⁶*raw count/sum (raw counts for allbarcodes in sample), which is referred to as prevalence values. Barcodeswith an average prevalence of 0.33 cpm per time point or less werediscarded. Full length construct-barcode associations obtained by SMRTsequencing (Pacific Biosciences) at select time points were used toidentify assemblies of interest. Incomplete constructs not having 3parts or ambiguous constructs having more than one construct call werefiltered out. Enrichment values for each assembly were calculated as(Normalized count at final time point+1)/(Normalized count at initialtime point+1). Candidate chimeric polypeptide genes were identified asconstructs with an enrichment above a threshold value specific for eachlibrary.

Results

Chimeric polypeptide candidates were designed to have 3 test domains,which included an extracellular and transmembrane domain (P1-2), a firstintracellular domain (P3), and a second intracellular domain (P4) (FIGS.30 and 31 ). Furthermore, the constructs included a DNA barcode to aidin analysis and identification of the construct using next-generationsequencing. Additionally, most constructs as shown in Table 6 includednucleic acid sequences encoding a recognition and/or elimination domainin frame with the extracellular and transmembrane domain. However, therecognition and/or elimination domains of those constructs encoding anextracellular or transmembrane fragment of LMP1 were intracellular forthose constructs that contained such a recognition and/or eliminationdomain. Library 1 constructs encoded a CAR upstream of the chimericpolypeptide candidate. A total of 226,840 conceptualized possiblecombinations were designed based on 20 differentextracellular-transmembrane domains, 106 possible P3 domains and 107possible P4 domains, one of which was a STOP codon. Not all of theconceptualized constructs were detected following viral vector assembly,lentiviral particle production, transduction of PMBC, and 7 days ofculture. For Library 1A, 57,815 constructs were present aftertransduction of PBMCs and 7 days of culture. For Library 2B, 69,578constructs were present after transduction of PBMCs and 7 days ofculture. Detailed information about the candidates analyzed, as well asdetailed results, are provided in Tables 7 to 11. Detailed informationabout the various candidate parts is provided in Table 6. The headersfor Tables 7-11 are as follows: BlockSequence is the code of P1-P2, P3,and P4 domains from Table 6 used in each construct; Norm_D7 is thenormalized counts at day 7; Enrich_DXX is the enrichment at day XXversus day 7, where XX is the day indicated on the table, for example,Enrich_D21 is the enrichment at day 21 versus day 7 and Enrich_D35 isthe enrichment at day 35 versus day 7. Data for a construct is shown tothe immediate right columns for the construct. Data for two constructsare shown in each row. For example, the first candidate identified inTable 7 is M008-S212-S075. For this candidate, the extracellular andtransmembrane domain (P1-2) is M008, the first intracellular domain (P3)is S212, and the second intracellular domain (P4) is S075. Detailedinformation about the identity of these modules, for example M008, isfound in Table 6. Table 6 discloses that M008 is LMP1 and that theconstruct includes a Myc tag. The amino acid sequences of the candidatedomains are provided in Table 6. For clarification, the GenBankidentifier in Table 6 provides a nucleic acid sequence that encodes apolypeptide domain. In some cases the nucleic acid sequence was modifiedto assist with cloning, or for codon optimization, but encodes the samepolypeptide as the GenBank sequence. In some cases, as will be apparentby the amino acid sequence provided in Table 6, a portion of thepolypeptide encoded by the GenBank sequence was used.

Candidate extracellular and transmembrane domains were selected fromknown mutant receptors such as cytokine or hormone receptors that havebeen reported to be constitutively active when expressed at least insome cell types. Ligand binding regions were not included in thecandidate extracellular and transmembrane domains. Mutations in suchreceptor mutants occur in the transmembrane region or juxtamembraneregion. Exemplary candidate extracellular and transmembrane domainsincluded IL7RA Ins PPCL, LMP1, CRLF2 F232C, CSF2RB V449E, CSF3R T640N,EPOR L251C 1252C, GHR E260C I270C, IL27RA F523C, and MPL S505N. Thecandidate extracellular and transmembrane domains included the wild typeviral protein LMP1, which is a multispan transmembrane protein that isknown to activate cell signaling independent of ligand when targeted tolipid rafts or when fused to CD40 (Kaykas et al. EMBO J. 20: 2641(2001)).

The identities of potential polypeptides chosen to occupy the first andsecond intracellular domains of the candidate chimeric polypeptides wereidentical. These candidate intracellular domains were intracellularsignaling domains of genes that are known in at least some cell types,to promote proliferation, survival (anti-apoptotic), and/or provide aco-stimulatory signal that enhances a differentiation state,proliferative potential or resistance to cell death. Some of theintracellular domains of candidate chimeric polypeptides are known toactivate JAK1/JAK2 signaling and STATS. Intracellular domains from someof the genes listed in Table 6 were included in the library. Exemplaryintracellular domains from these genes are provided in the P3 and P4locations of Tables 7 to 11. Detailed information about these P3 and P4domains are provided in Table 6.

For Library 1A, which included constructs that encoded the chimericpolypeptides and a CAR, 172 top candidate chimeric polypeptides wereidentified (See Table 7, in which the enrichment was calculated as Log2(Normalized count at the last day indicated on the table+1)−log2(Normalized count at day 7+1)) that promoted PBMC proliferation to thegreatest magnitude (and likely survival too) between day 7 and the lastday indicated on the table when cultured in the absence of IL-2. It isnoteworthy that this and all of the screening experiments of Examples 17and 18 are competitive experiments. Thus, a negative result does notmean that a construct does not promote proliferation of T cells in theabsence of growth factors such as IL-2. It only means that relative toother constructs tested, it was outperformed with respect toproliferation.

For Library 1A, some of the chimeric polypeptide constructs promotedcell proliferation between day 7 and the last day indicated on the tablefor every extracellular and transmembrane mutant domain tested in theP1-2 position. Furthermore, when all results from all constructs derivedfrom the same position P1-2 mutant were combined, all extracellular andtransmembrane domains tested yielded cell enrichment of greater than 1(i.e. promoted cell proliferation) between day 7 and the last dayindicated on the table. Examples of extracellular and/or transmembranedomains or portions and/or mutants thereof, that were present inconstructs that promoted cell proliferation are provided in Table 7.Examples of extracellular and/or transmembrane domains or portionsand/or mutants thereof, that were present in constructs in the repeatedscreen referred to as Library 1.1A that promoted cell proliferation areprovided in Table 9. Examples of extracellular and/or transmembranedomains or portions and/or mutants thereof, that were present inconstructs in the repeated screen referred to as Library 1.1B, whichincluded cells transduced with Library 1 that were not fed PBMCs, thatpromoted cell proliferation are provided in Table 10.

For Library 1A, intracellular domains from CD3D, CD3E, CD8A, CD27, CD40,CD79B, IFNAR1, IL2RA, IL3RA, IL13RA2, TNFRSF8, and TNFRSF9, or mutantsthereof that are known to have signaling activity if such mutants arepresent in the constructs provided in the tables, when found at thefirst intracellular domain position (P3) of candidate chimericpolypeptides promoted proliferation of PBMCs between day 7 and the lastday indicated on the table, when data was considered in a combinedmanner for all constructs with first intracellular domains (P3) derivedfrom that gene. This conclusion is based on enrichment of sequencecounts of constructs in mixed cultured PBMC cell populations, such thatat least 3 times more counts for such domain were present at the lastday indicated on the table than day 7 for Library 1A, when results forall constructs for a gene are combined. Examples of first intracellulardomains or portions and/or mutants thereof, that were present inconstructs that promoted the highest magnitude of cell proliferation areprovided in Table 7. Examples of first intracellular domains or portionsand/or mutants thereof, that were present in constructs in the repeatedscreen referred to as Library 1.1A that promoted the highest magnitudeof cell proliferation are provided in Table 9. Examples of firstintracellular domains or portions and/or mutants thereof, that werepresent in constructs in the repeated screen referred to as Library1.1B, which included cells transduced with Library 1 that were not fedPBMCs, that promoted cell proliferation are provided in Table 10.

For Library 1A, intracellular domains from CD3D, CD3G, CD8A, CD8B, CD27,CD40, CD79B, CRLF2, FCGR2C, ICOS, IL2RA, IL13RA1, IL13RA2, IL15RA,TNFRSF9, and TNFRSF18, or mutants thereof that are known to havesignaling activity if such mutants are present in the tables, when foundat the second intracellular domain position (P4) of candidate chimericpolypeptides promoted proliferation of PBMCs between day 7 and the lastday indicated on the table, when data was considered in a combinedmanner for all constructs with a second intracellular domains (P4)derived from that gene. This conclusion is based on enrichment ofsequence counts of constructs in mixed cultured PBMC cell populations,such that at least 2.9 times more counts for such domain were present atday 35 than day 7 for Library 1A, when results for all constructs for agene are combined. Examples of second intracellular domains or portionsand/or mutants thereof, that were present in constructs that promotedthe highest magnitude of cell proliferation are provided in Table 7.Examples of second intracellular domains or portions and/or mutantsthereof, that were present in constructs in the repeated screen referredto as Library 1.1A that promoted cell proliferation are provided inTable 9. Examples of second intracellular domains or portions and/ormutants thereof, that were present in constructs in the repeated screenreferred to as Library 1.1B, which included cells transduced withLibrary 1 that were not fed PBMCs, that promoted cell proliferation areprovided in Table 10.

More specifically for Library 1A, of the 5,996 constructs with MPL S505N(MPL proto-oncogene, thrombopoietin receptor with serine 505 mutated toasparagine) as the P1-P2 module, 165 were positive, which was defined asconstructs having an enrichment above two for Library 1A. Of the 436constructs with CD3D (CD3d molecule) as the P3 module, 24 were positive.Of the 528 constructs with CD3E (CD3e molecule) as the P3 module, 17were positive. Of the 261 constructs with CD8A (CD8a molecule) as the P3module, 14 were positive. Of the 1,022 constructs with CD40 (CD40molecule) as the P3 module, 50 were positive. Of the 568 constructs withIL3RA (interleukin 3 receptor subunit alpha) as the P3 module, 26 werepositive. Of the 556 constructs with CD79B (CD79b molecule) as the P3module, 27 were positive. Of the 131 constructs with IFNAR1 (interferonalpha and beta receptor subunit 1) as the P3 module, 7 were positive. Ofthe 519 constructs with IL13RA2 (interleukin 13 receptor subunit alpha2) as the P3 module, 33 were positive. Of the 571 constructs with IL2RA(interleukin 2 receptor subunit alpha) as the P3 module, 28 werepositive. Of the 618 constructs with CD27 (CD27 molecule) as the P3module, 30 were positive. Of the 573 constructs with TNFRSF8 (TNFreceptor superfamily member 8) as the P3 module, 15 were positive. Ofthe 573 constructs with TNFRSF9 (TNF receptor superfamily member 9) asthe P3 module, 25 were positive. Of the 360 constructs with IL13RA2(interleukin 13 receptor subunit alpha 2) as the P4 module, 25 werepositive. Of the 490 constructs with IL2RA (interleukin 2 receptorsubunit alpha) as the P4 module, 28 were positive. Of the 489 constructswith IL15RA (interleukin 15 receptor subunit alpha) as the P4 module, 22were positive. Of the 590 constructs with CD8A (CD8a molecule) as the P4module, 25 were positive. Of the 619 constructs with IL13RA1(interleukin 13 receptor subunit alpha 1) as the P4 module, 25 werepositive. Of the 637 constructs with CD3G (CD3g molecule) as the P4module, 30 were positive. Of the 618 constructs with CD3D (CD3dmolecule) as the P4 module, 37 were positive. Of the 673 constructs withCD27 (CD27 molecule) as the P4 module, 29 were positive. Of the 654constructs with CD79B (CD79b molecule) as the P4 module, 33 werepositive. Of the 728 constructs with TNFRSF9 (TNF receptor superfamilymember 9) as the P4 module, 30 were positive. Of the 1,169 constructswith CD40 (CD40 molecule) as the P4 module, 53 were positive. Of the1,324 constructs with TNFRSF18 (TNF receptor superfamily member 18) asthe P4 module, 54 were positive. Of the 1,739 constructs with CD8B asthe P4 module, 73 were positive. Of the 286 constructs with CRLF2 as theP4 module, 8 were positive. Of the 725 constructs with FCGR2C as the P4module, 29 were positive. Of the 602 constructs with ICOS as the P4module, 24 were positive.

For Libraries 1A and 1.1A, the constructs M001-S116-S044,M024-S192-S045, M001-S047-S102, M048-S195-S043, M012-S216-S211, andM030-S170-S194 had particularly noteworthy enrichments in both screens.For the purposes of the repeated screens, constructs with particularlynoteworthy enrichments were those that had a log₂((normalized count dataon the last day+1)/(normalized count data on day 7+1)) value above 2.

Further information about the first and second intracellular domains inconstructs with particularly noteworthy enrichments in screens of bothLibrary 1A and Library 1.1A is provided in Table 19, including thegene(s) the first and second intracellular domain are derived from,whether the first and/or second intracellular domain are cytokinereceptors, and whether the first and/or second intracellular domain haveat least one ITAM motif. Constructs with intracellular domains fromIL6R, MYD88, CD27, MYD88, TNFRSF18, or IL31RA, when present at the firstintracellular domain (P3), and constructs with intracellular domainsfrom CD8B, IL1RL1, CD8A, TNFRSF4, or MYD88, when present at the secondintracellular domain (P4), showed particularly noteworthy enrichments inboth Libraries 1A and 1.1A (Table 19). Constructs with domains from thecytokine receptors IL6R, CD27, TNFRSF18, or IL31RA, when present at thefirst intracellular domain (P3), and constructs with domains from thecytokine receptors ILIRL1 or TNFRSF4, when present at the secondintracellular domain (P4), showed particularly noteworthy enrichments inboth Libraries 1A and 1.1A (Table 19).

In Library 2B, which included constructs that encoded the chimericpolypeptides and did not encode a CAR, 167 top candidates wereidentified (See Table 8, in which the enrichment was calculated as Log2(Normalized count at the last day indicated on the table+1)−log2(Normalized count at day 7+1)) that promoted PBMC proliferation betweenday 7 and the last day indicated on the table when cultured in theabsence of IL-2. It is noteworthy that, in general enrichment was lessthan that found for Library 1A constructs, which included a CAR.

For Library 2B, CSF3R T640N in the P1-2 position, promoted cellproliferation between day 7 and the last day indicated on the tablebetter than other extracellular and transmembrane (P1-2) domains tested,when all results from all constructs derived from an extracellular andtransmembrane domain were combined, yielding an enrichment factor ofgreater than 2.5. Examples of extracellular and/or transmembrane domainsor portions and/or mutants thereof, that were present in constructs thatpromoted the highest magnitude of cell proliferation are provided inTable 8. Examples of extracellular and/or transmembrane domains orportions and/or mutants thereof, that were present in constructs in therepeated screen referred to as Library 2.1B that promoted the highestmagnitude of cell proliferation are provided in Table 11.

For Library 2B, intracellular domains from CD8A, CD40, IFNAR1, IL31RA,and MyD88, or mutants thereof that are known to have signaling activityif such mutants are present in the tables, when found at the firstintracellular domain position (P3) of candidate chimeric polypeptidespromoted cell proliferation of PBMCs between day 7 and the last dayindicated on the table, when data is considered in a combined manner forall constructs with first intracellular domains (P3) derived from thatgene. This conclusion is based on enrichment of sequence counts ofconstructs in mixed cultured PBMC cell populations, such that at least 2times more counts for such domain were present at day 28 than day 7 forLibrary 2B, when results for all constructs for a gene are combined.Examples of first intracellular domains or portions and/or mutantsthereof, that were present in constructs that promoted the largestmagnitude of cell proliferation are provided in Table 8. Examples offirst intracellular domains or portions and/or mutants thereof, thatwere present in constructs in the repeated screen referred to as Library2.1B that promoted the highest magnitude of cell proliferation areprovided in Table 11.

For Library 2B, intracellular domains from CD27, or mutants thereof thatare known to have signaling activity if such mutants are present in theconstructs provided in the tables, when found at the secondintracellular domain position (P4) of chimeric polypeptide candidates,when data is considered in a combined manner for all constructs with asecond intracellular domains (P4) derived from that gene, promotedproliferation of PBMCs between day 7 and the last day indicated on thetable. This conclusion is based on enrichment of sequence counts ofconstructs in mixed cultured PBMC cell populations, such that 11 timesmore counts for such domain were present at day 28 than day 7 forLibrary 2B, when results for all constructs for a gene are combined.Examples of second intracellular domains or portions and/or mutantsthereof, that were present in constructs that promoted the largestmagnitude of cell proliferation are provided in Table 8. Examples ofsecond intracellular domains or portions and/or mutants thereof, thatwere present in constructs in the repeated screen referred to as Library2.1B that promoted the largest magnitude of cell proliferation areprovided in Table 11.

More specifically, for Library 2B, of the 6,909 constructs with CSF3RT640N (colony stimulating factor 3 receptor with threonine 640 mutatedto asparagine) as the P1/P2 module for Library 2B, 59 were positive,which was defined as constructs having an enrichment above two forLibrary 2B. Of the 184 constructs with IFNAR1 (interferon alpha and betareceptor subunit 1) as the P3 module for Library 2B, 3 were positive. Ofthe 1,258 constructs with CD40 (CD40 molecule) as the P3 module forLibrary 2B, 17 were positive. Of the 851 constructs with CD27 (CD27molecule) as the P4 module for Library 2B, 11 were positive. Of the 418constructs with CD8A as the P3 module for Library 2B, 10 were positive.Of the 1,385 constructs with IL31RA as the P3 module for Library 2B, 12were positive. Of the 5,757 constructs with MyD88 as the P3 module forLibrary 2B, 56 were positive.

For Libraries 2B and 2.1B, the constructs M007-S049-S051,M007-S050-S039, M012-S050-S043, M012-S161-S213, M030-S142-S049,M001-S145-S130, M018-S085-S039, M018-S075-S053, M012-S135-S074, andM007-S214-S077 had particularly noteworthy enrichments in both screens.For the purposes of the repeated screens, constructs with particularlynoteworthy enrichments were those that had a log₂((normalized count dataon the last day+1)/(normalized count data on day 7+1)) value above 2.

Further information about the first and second intracellular domains inconstructs with particularly noteworthy enrichments in screens of bothLibrary 2B and Library 2.1B is provided in Table 20, including thegene(s) the first and second intracellular domain are derived from,whether the first and/or second intracellular domain are cytokinereceptors, and whether the first and/or second intracellular domain haveat least one ITAM motif. Constructs with intracellular domains fromCD28, CD40, IL22RA1, IL13RA2, IL17RB, IFNGR2, FCGR2C, IL11RA, orTNFRSF14, when present at the first intracellular domain (P3), andconstructs with intracellular domains from CD40, CD3G, CD8A, TNFRSF9,CD28, IL10RB, CD79B, FCER1G, or GHR, when present at the secondintracellular domain (P4), showed particularly noteworthy enrichments inboth Libraries 2B and 2.1B (Table 20). Constructs with domains from thecytokine receptors CD40, IL22RA1, IL13RA2, IL17RB, IFNGR2, FCGR2C,IL11RA, or TNFRSF14, when present at the first intracellular domain(P3), and constructs with domains from the cytokine receptors TNFRSF9,IL10RB, FCER1G, GHR, or CD40, when present at the second intracellulardomain (P4), showed particularly noteworthy enrichments in bothLibraries 2B and 2.1B (Table 20). Constructs with domains containingITAM motifs from FCGR2C, when present at the first intracellular domain(P3), and constructs with domains containing ITAM motifs from CD3G,CD79B, or FCER1G, when present at the second intracellular domain (P4),showed particularly noteworthy enrichments in both Libraries 2B and 2.1B(Table 20).

Example 18. Identification of Candidate Chimeric PolypeptideLymphoproliferative Elements

In this example, two chimeric polypeptide libraries (Library 3 andLibrary 4) of candidate (putative) chimeric lymphoproliferative elementswere assembled into viral vectors from pools ofextracellular-transmembrane block sequences, intracellular blocksequences, and a barcode library according to the chimericpolypeptide-encoding construct provided in FIGS. 32 and 33 . Theproliferation of library-transduced PBMCs was followed over time:genomic DNA was extracted from PBMCs at one-week intervals and thebarcode region amplified for DNA sequencing to estimate the number ofcells in these mixed cultures of PBMCs containing each of the testcandidate chimeric polypeptides. Thus, the screen tested the ability ofthe candidate chimeric polypeptides to promote PBMC cell proliferationin the absence of exogenously added IL-2 or any other exogenouscytokine.

Two libraries were made and analyzed in this study: Library 3 (which wasused in the screens identified as Libraries 3A, 3B, 3.1A, and 3.1B) wasflanked with a CAR at the 5′ end of the chimeric polypeptide, whileLibrary 4 (which was used in the screens identified as Libraries 4B and4.1B) did not include a CAR. FIG. 32 provides a schematic of anon-limiting, exemplary transgene expression cassette containing apolynucleotide sequence encoding a CAR and a candidate CLE of Library 3driven by an EF-1 alpha promoter and a Kozak-type sequence(GCCGCCACC(SEQ ID NO:519)), in a lentiviral vector backbone. The CAR wasFLAG tagged and contained an ASTR directed to CD19, a stalk andtransmembrane portion of CD8, and an intracellular activating domainfrom CD3z. Each candidate lymphoproliferative element of Library 3included 4 modules; an extracellular module (P1), a transmembrane module(P2), and 2 intracellular modules (P3 and P4). The P1 module alsoencoded a Myc recognition and/or elimination domain. A triple stopsequence (TAATAGTGA (SEQ ID NO:520)) separated P4 from a DNA barcode(P5). A WPRE(GTCCTTTCCATGGCGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCCCGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCCGATCTCCCTTTGGGCCGCCTCCCCCCC TG (SEQID NO:521)) was present between the last stop codon (starting 4 bp afterthe last nucleotide of the last stop codon) and the 3′ LTR (whichstarted 83 nucleotides after the last nucleotide of the WPRE).

The CAR and P1 of Library 3 were separated by a polynucleotide sequenceencoding a T2A ribosomal skip sequence.

FIG. 33 provides a schematic of a non-limiting, exemplary transgeneexpression cassette containing a polynucleotide sequence a candidate CLEof Library 4 driven by an EF-1 alpha promoter and a Kozak-type sequence(GCCGCCACC(SEQ ID NO:519)), in a lentiviral vector backbone. Eachcandidate lymphoproliferative element of Library 4 included 4 modules;an extracellular module (P1), a transmembrane module (P2), and 2intracellular modules (P3 and P4). The P1 module also encoded a Mycrecognition and/or elimination domain. A triple stop sequence (TAATAGTGA(SEQ ID NO:520)) separated P4 from a DNA barcode (P5). A WPRE(GTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCC TG SEQID NO:521)) was present between the last stop codon (starting 4 bp afterthe last nucleotide of the last stop codon) and the 3′ LTR (whichstarted 83 nucleotides after the last nucleotide of the WPRE).

The assembled chimeric polypeptide parts of both libraries followed theexact same design with 4 positions (1 extracellular module (P1), 1transmembrane module (P2), and 2 intracellular parts (P3 and P4), one ofwhich could be an early termination signal) (FIGS. 32 and 33 ). Nucleicacids encoding full or variant versions of either a Myc tag or an eTAGrecognition and/or elimination domain (referred to in Table 6 as “eTAG”or “Myc Tag”) was inserted at the 5′ terminus of the CAR-encodingsequences in Library 3 and at the 5′ terminus of the c-Jun domain in P1in Library 4. A signal sequence was inserted at the 5′ terminus of theCAR-encoding sequences in Library 3 and at the 5′ terminus of therecognition and/or elimination domain-encoding sequences of Library 4.The general design and construction of the library, including thebarcode, was as disclosed in Example 17 herein, except for the P1 and P2domains, as set out in more detail later in this Example. Furthermore,the clearance domain was on some constructs, as indicated in Table 6,and when present was an eTag variant or a Myc tag.

Synthesis of Viral Vectors and Lentiviral Production

Vectors were synthesized and lentiviral particles were produced for eachlibrary as disclosed in Example 17.

Transduction and Culturing of PBMCs

For Library 3A, whole human blood from a healthy donor (89.1 ml) wascollected into a 200 ml blood bag containing CDP anticoagulant (Nanger;5-200). The blood was processed using density gradient centrifugationwith Ficoll-Pacque™ (General Electric) using a Sepax 2 S-100 device(Biosafe; 14000) following manufacturer's instruction and kit CS900.2(BioSafe; 1008), to obtain peripheral blood mononuclear cells (PBMCs).Yield of viable PBMC was 8.4×10⁷ cells. Ten vials of cells at 5×10⁶ PBMCper vial were frozen for later use in feeding CD19+ B cells for CD19-CARcontaining Library 3A samples. 3×10⁷ PBMC were added to one G-Rex 100Mcell culture device (Wilson Wolf, 81100S) to a final concentration of0.5×10⁶ viable cells/ml in a final volume of 60 ml of Complete OpTmizer™CTS™ T-Cell Expansion SFM (OpTmizer™ CTS™ T-Cell Expansion Basal Medium1 L (Thermo Fisher, A10221) supplemented with ml OpTmizer™ CTS™ T-CellExpansion Supplement (Thermo Fisher, A10484-02), CTS™ Immune Cell SR(Thermo Fisher, A2596101), and CTS™ GlutaMAX™-I Supplement (ThermoFisher, A1286001) according to manufacturer's instructions. Recombinanthuman interleukin-2 (IL-2) (Novoprotein) was also added to aconcentration of 100 IU/ml along with activating anti-CD3 Ab (OKT3,Novoprotein) added at a concentration of 50 ng/ml, to activate the PBMCsfor viral transduction. The G-Rex device was incubated overnight in astandard humidified tissue culture incubator at 37° C. and 5% CO₂. Afterovernight incubation, lentivirus particle preparations containing thedesired test chimeric polypeptide-encoding constructs (Library 3) wereadded to the G-Rex device at a multiplicity of infection (MOI) of 5. TheG-Rex device was incubated overnight in a standard humidified tissueculture incubator at 37° C. and 5% CO₂. Following the overnightincubation, additional Complete OpTmizer™ CTS™ T-Cell Expansion SFM wasadded to bring the final volume to 500 ml. No additional IL-2 or otherexogenous cytokine was added at this or following cell culture steps.The G-Rex® device was incubated in a standard humidified tissue cultureincubator at 37° C. and 5% CO₂ for 7 days from PBMC isolation. On day 7,the cells from the G-Rex device transduced with Library 3 were collectedby centrifugation and resuspended into fresh Complete OpTmizer™ CTS™T-Cell Expansion SFM without IL-2. 5×10⁷ of Library 3 cells were placedin a new G-Rex 100M device containing 500 ml Complete OpTmizer™ CTS™T-Cell Expansion SFM without IL-2 and labelled as Library 3A. 1.1×10⁸ ofLibrary 3 cells were placed in a new G-Rex 100M device containing 500 mlComplete OpTmizer™ CTS™ T-Cell Expansion SFM without IL-2 and labelledas Library 3B. In these screens, cells fed with PBMCs were designatedwith an A after the library number, for example screens performed withcells transduced with Library 3 and fed with PBMCs are referred to asLibrary 3A herein. Four vials of thawed Day 0 frozen donor matched PBMC(2×10⁷ PBMC) was added to the G-Rex devices containing cells transducedwith library 3A to provide CD19+ B-cell activation of the CD19 ASTR.Library 3B received no Day 0 PBMC. In these screens, cells not fed withPBMCs were designated with a B after the library number. For examplescreens performed with cells transduced with Library 3 and not fed withPBMCs are referred to as Library 3B herein. The G-Rex devices wereincubated in a standard humidified tissue culture incubator at 37° C.and 5% CO₂. On day 14, the cells from the two G-Rex devices (library 3A,library 3B) were collected by centrifugation and resuspended into freshComplete OpTmizer™ CTS™ T-Cell Expansion SFM without IL-2. These wereeach placed into individual wells of 6-well G-Rex plates (Wilson-Wolf;80240M) containing 30 ml Complete OpTmizer™ CTS™ T-Cell Expansion SFMwithout IL-2. One vial of thawed Day 0 frozen donor matched PBMC (5×10⁶PBMC) was added to the library 3A G-Rex to provide CD19+ B-cellactivation of the CD19 ASTR. The G-Rex devices were incubated in astandard humidified tissue culture incubator at 37° C. and 5% CO₂. Thisprocess was repeated on Day 21, Day 28, Day 35, and Day 42 except twovials of frozen Day 0 PBMC were added to Library 3A at day 21, and onevial on Days 28, 35, and 42. Library 3B was not cultured past day 21.These screens were repeated, except the Myc tag on the P1 part wasreplaced with an eTag as shown in Tables 6, 14, and 15, and designatedas Libraries 3.1A and 3.1B, where the A and B indicated whether or notthe cells were fed with PBMCs as discussed above.

Cells from Library 3A were prepared for analysis by flow cytometry onDay 49. Cells were harvested by centrifugation (400 g, 5 minutes) andlayered over Ficoll (Ficoll-Paque Premium (GE, cat #17-5442-02)according to manufacturer's directions. 1.7×10⁶ cells were resuspendedin 450 μl FACs Staining Buffer (554656, BD). Resuspended cells werealiquoted into 9 eppendorf tubes, each containing 50 μl or 1.8×10⁵cells. 2.5 μl human Fc Block (564220, BD) was added into each tube andincubated at room temperature for 10 minutes. The following antibodyrecipe was added into the 50 μl samples to stain for CD3, CD4, CD8,CD56, CD19, FLAG Tag (CAR+): 2.5 μl anti-CD3-BV421 antibody (cloneOKT3,317344, Biolegend), 2.5 μl anti-CD8-BV510 antibody (clone RPA-T8(301048, Biolegend), 2.5 μl anti-CD4-PE-Cy7 antibody (clone OKT4(317412, Biolegend), 2.5 μl anti-CD56-BV785(362550, Biolegend) and 0.5μl PE anti-FLAG Tag (anti-DYKDDDDK, Clone L5, 637310, Biolegend) for 5color stained samples. 2.5 μl BB515 CD19 antibody (clone HIB19, 564456,BD) and 0.5 ul PE anti-FLAG Tag antibody were added for 2 color stainingsamples. Samples single stained for CD3, CD4, CD8, CD56, or FLAG Tagwere also prepared using the antibodies and volumes above. 2.5 μl BV421IgG (mouse IgG2a,400259, Biolegend), 2.5 μl PE/CY5 IgG (mouse IgG2b,400317, Biolegend), 2.5 μl BV510 IgG (mouse IgG1, 400171, Biolegend),2.5 μl BV785 IgG (mouse IgG1, 400169, Biolegend) and 10 μl PE IgG (mouseIgG1, 555749, BD) were added for IgG control stained samples. Noantibody was added for a non-stained control. Samples were incubated 30minutes, on ice. The samples were centrifuged (400 g, 5 minutes) andsupernatant was removed. The samples were washed with 0.5 ml FACSStaining Buffer twice. The stained cell pellets were resuspended in 125μl FACS Staining Buffer and then fixed by adding 125 ul BD Fixativebuffer (554655 BD). The samples were incubated with fixation buffer for15 minutes at 4° C. The samples were washed twice with 500 μl FACSStaining Buffer and resuspended with 0.4 ml FACS Staining Buffer. FACsanalysis of samples was performed on a Novocyte flow cytometer (ACEABiosciences) and analyzed using a lymphocyte gate based on forward andside scatter.

For Library 4B, whole human blood from a healthy donor (70 ml) wascollected into a 200 ml blood bag containing CDP anticoagulant (Nanger;S-200). The blood was processed using density gradient centrifugationwith Ficoll-Pacque™ (General Electric) using a Sepax 2 S-100 device(Biosafe; 14000) following manufacturer's instruction and kit CS900.2(BioSafe; 1008), to obtain peripheral blood mononuclear cells (PBMCs).Yield of viable PBMC was 4.9×10⁷ cells. 4.9×10⁷ PBMC were added to oneG-Rex 100M cell culture device (Wilson Wolf, 81100S) to a finalconcentration of 0.5×10⁶ viable cells/ml in a final volume of 98 ml ofComplete OpTmizer™ CTS™ T-Cell Expansion SFM. Recombinant humaninterleukin-2 (IL-2) (Novoprotein) was also added to a concentration of100 IU/ml along with activating anti-CD3 Ab (OKT3, Novoprotein) added ata concentration of 50 ng/ml, to activate the PBMC for viraltransduction. The G-Rex device was incubated overnight in a standardhumidified tissue culture incubator at 37° C. and 5% CO₂. Afterovernight incubation, lentivirus particle preparations containing thedesired test chimeric polypeptide-encoding constructs (Library 4) wereadded to the G-Rex device at a multiplicity of infection (MOI) of 5. TheG-Rex device was incubated overnight in a standard humidified tissueculture incubator at 37° C. and 5% CO₂. Following the overnightincubation, additional Complete OpTmizer™ CTS™ T-Cell Expansion SFM wasadded to bring the final volume to 500 ml. No additional IL-2 was addedat this or following cell culture steps. The G-Rex® device was incubatedin a standard humidified tissue culture incubator at 37° C. and 5% CO₂for 7 days from PBMC isolation. On day 7, the cells from the G-Rexdevice transduced with library 4 were collected by centrifugation andresuspended into fresh Complete OpTmizer™ CTS™ T-Cell Expansion SFMwithout IL-2. 1.63×10⁸ of Library 4 cells were placed in a new G-Rex100M device containing 500 ml Complete OpTmizer™ CTS™ T-Cell ExpansionSFM without IL-2 and labelled as Library 4. Library 4 received no Day 0PBMC. Thus, In these screens, the libraries were designated with a Bafter the library number, for example screens performed with cellstransduced with Library 4 were not fed with PBMCs and are referred to asLibrary 4B herein. The G-Rex device was incubated in a standardhumidified tissue culture incubator at 37° C. and 5% CO₂. On day 14, thecells from the G-Rex device (Library 4) were collected by centrifugationand resuspended into fresh Complete OpTmizer™ CTS™ T-Cell Expansion SFMwithout IL-2. These cells were each placed into an individual well of6-well G-rex plates (Wilson-Wolf; 80240M) containing 30 ml CompleteOpTmizer™ CTS™ T-Cell Expansion SFM without IL-2. The G-Rex device wasincubated in a standard humidified tissue culture incubator at 37° C.and 5% CO₂. Library 4 samples were not cultured past Day 21. Thesescreens were repeated, except the Myc tag on the P1 part was replacedwith an eTag as shown in Tables 6 and 16, and designated as Library4.1B, where the B indicated the cells were not fed with PBMCs asdiscussed above.

Samples (7×10⁴ to ˜4-8×10⁶ cells) were collected at various dayspost-transduction (Days 7 and 21) and genomic DNA was purified usingGenElute™ Mammalian Genomic DNA Miniprep following the kits protocol. Insome cases, samples were purified using density gradient centrifugationwith Ficoll-Pacque™ (General Electric) to remove dead cells prior togenomic DNA extraction. Purified genomic DNA was sequenced using anIllumina HiSeq, generating paired-end 150 bp reads. Usually, a subset of10 million reads was extracted from each indexed fastq file andprocessed for analysis using barcode_reader, a custom R scriptengineered to extract barcode sequences based on the presence of aconstant region. Purified genomic DNA was also sequenced on a PacBiosequencing system to associate barcodes with constructs.

Data Analysis

Data analysis was performed as disclosed in Example 17 except the timepoints differed.

Results

In this experiment, chimeric polypeptide candidates were designed tohave 4 test domains, which included an extracellular domain (P1), atransmembrane domain (P2), a first intracellular domain (P3), and asecond intracellular domain (P4) (FIGS. 32 and 33 ). As explained inExample 17, the constructs included a DNA barcode to aid in analysis andidentification of the construct using next-generation sequencing.Additionally, all of the constructs included nucleic acid sequencesencoding a recognition and/or elimination domain in frame with theextracellular domain. Constructs in Library 3, but not Library 4.encoded a CAR upstream of the chimeric polypeptide candidate, which wasidentical in structure to the CAR used in Library 1 (Example 17).

The extracellular domain used for Library 3 and Library 4 was a variantof c-Jun that included the leucine zipper motif (NM_002228_3) known tobe a dimerizing motif (see, e.g. Chinenov and Kerppola, Oncogene. 2001Apr. 30; 20(19):2438-52). A spacer of between 0 and 4 alanine residueswas included in the candidate chimeric polypeptides between the c-Junextracellular domain and the transmembrane domain. Not to be limited bytheory, it is believed that the alanine spacer could affect signaling ofintracellular domains connected to the leucine zipper extracellularregion via the transmembrane domain, by changing the orientation of theconnected transmembrane domains and/or the intracellular domains.

Candidate transmembrane domains were selected from known single spanningtransmembrane domains from wild-type and mutant Type I receptors such ascytokine, hormone, costimulatory, or activating receptors that have beenreported to be constitutively active when expressed at least in somecell types. Mutations in such receptor mutants selected for Library 3and Library 4 occur in the transmembrane region. Exemplary transmembranedomains for Libraries 3A, 3B, 3.1A, 3.1B, 4B, and 4.1B can be identifiedin Tables 12 to 17. The headers for Tables 12-17 are as follows:BlockSequence is the code of P1, P2, P3, and P4 domains from Table 6used in each construct; Norm_D7 is the normalized counts at day 7;Enrich_DXX is the enrichment at day XX versus day 7, where XX is the dayindicated on the table, for example, Enrich_D21 is the enrichment at day21 versus day 7 and Enrich_D35 is the enrichment at day 35 versus day 7.Data for a construct is shown to the immediate right columns for theconstruct. Data for two constructs are shown in each row. For example,the first construct that is identified in Table 12 isE013-T047-S158-S080, which sets out P1-P2-P3-P4. In that construct, thetransmembrane domain (P2) is T047, which is the transmembrane domain ofIL7RA Ins PPCL (interleukin 7 receptor), as provided in Table 6, alongwith the amino acid sequence of this domain that was included in thisconstruct. The same first intracellular domains (P3) and secondintracellular domains (P4) included in Example 17 were included in thelibraries in this example except that one of the P3 parts was a spacer(see below).

A total of 5 conceptualized possible combinations were designed based ona Jun leucine zipper extracellular domain with a 0-4 alanine spacerseparating it from 82 different transmembrane domains, 81 possible P3domains, one of which was a 23 amino acid spacer, and 21 possible P4domains, one of which was a STOP codon. Not all of the conceptualizedconstructs were detected following viral vector assembly, lentiviralparticle production, transduction of PMBC, and 7 days of culture. ForLibraries 3A and 3B, 68,951 constructs were present after transductionof PBMCs and 7 days of culture. For Library 4B, 50,652 constructs werepresent after transduction of PBMCs and 7 days of culture. Detailedinformation about the candidates analyzed can be determined from Tables6 and 12 to 17. The coding system for constructs is the same asexplained for Example 17, as set out in the preceding paragraph.

For Libraries 3A, 3B, 3.1A, 3.1B, 4B, and 4,1B, constructs wereidentified that promoted PBMC cell proliferation between day 7 and thelast day indicated on the table. Certain polypeptides in specificmodules of the CLEs were among the top hits across the libraries 3A, 3B,and 4. These CLEs promoted proliferation to the largest magnitude. Forexample, exemplary constructs containing CD40 or ICOS in the P2position; MPL, MyD88, LEPR, or IFNAR2 in the P3 position; and/or CD40,CD79B, or CD27 in the P4 position were among the top 5 most frequentchimeric polypeptides in at least 2 of the 3 Libraries 3A, 3B, and 4B(see Tables 12, 13, and 16). Strikingly, MPL was the most frequentchimeric polypeptide at P3 by a large margin for all 3 libraries. Notethe top hits from P3 (MPL, MyD88, LEPR, and IFNAR2) were not included inthe P4 position for these constructs.

In Libraries 3A and 3B, which included constructs that encoded thechimeric polypeptides and a CAR and transduced PBMCs that weresupplemented with (Library 3A) or without (Library 3B) freshuntransduced PBMCs, 126 and 127 top candidates were identified,respectively (See Tables 12 and 13) that promoted PBMC proliferationbetween day 7 and the last day indicated on the table when cultured inthe absence of IL-2. The top candidate lists of Tables 12 and 13 weregenerated by combining the top 100 most prevalent candidates at day 21based on initial interim data analysis, and the top 100 most enrichedcandidate constructs between day 21 and day 7 based on the initialinterim data analysis. The initial interim data was generated bydecoding part of the coded library such that 66 of the top 100constructs, and 538 out of the top 1000 constructs had been decoded forlibrary 3A; and 77 out of the top 100 constructs, and 464 out of the top1000 constructs had been decoded for library 3B.

For Library 3A, transmembrane domains (P2) from CD40, CD8B, CRLF2,CSF2RA, FCGR2C, ICOS, IFNAR1, IFNGR1, IL10RB, IL18R1, IL18RAP, IL3RA,LEPR, and PRLR, or mutants thereof that are known to promote signalingactivity in certain cell types if such mutants are present in theconstructs provided in the tables, promoted proliferation of PBMCsbetween day 7 and day 21, when data is considered in a combined mannerfor all constructs with a transmembrane domain derived from that gene(data not shown). This conclusion is based on enrichment of sequencecounts of constructs in mixed cultured PBMC cell populations, such thatenrichment, calculated as the logarithm in base 2 of the ratio betweennormalized count at day 21 plus one and normalized count at day 7 plusone, was at least 2 for Library 3A, when results for all constructs fora gene are combined. For Library 3B, transmembrane domains from CD40,ICOS, CSF2RA, IL18R1, IL3RA, and TNFRSF14, were the best performers whenanalyzed in this way. Examples of transmembrane domains or portionsand/or mutants thereof, that were present in constructs that promotedcell proliferation for Libraries 3A and 3B on day 35 and 21,respectively, are provided in Tables 12 and 13. Examples oftransmembrane domains or portions and/or mutants thereof, that werepresent in constructs in the repeated screens referred to as Libraries3.1A and 3.1B that promoted cell proliferation are provided in Table 14and 15.

For Library 3A, first intracellular (P3) domains from IL17RD, IL17RE,ILIRAP, IL23R, and MPL, or mutants thereof that are known to promotesignaling activity in certain cell types if such mutants are present inthe constructs provided in the tables, when found at the firstintracellular domain (P3) of candidate chimeric polypeptides elementsherein, promoted proliferation of PBMCs between day 7 and day 21, whendata is considered in a combined manner for all constructs with a firstintracellular domain derived from that gene (data not shown). Thisconclusion is based on enrichment of sequence counts of constructs inmixed cultured PBMC cell populations, such that enrichment, calculatedas the logarithm in base 2 of the ratio between normalized count at day21 plus one and normalized count at day 7 plus one, was at least 2 forLibrary 3A, when results for all constructs for a gene are combined. ForLibrary 3B, first intracellular domains from IL21R, MPL, and IL2RB, ormutants thereof that are known to promote signaling activity in certaincell types if such mutants are present in the tables, were the bestperformers when analyzed in this way. Examples of first intracellulardomains or portions and/or mutants thereof that were present inconstructs that promoted the largest magnitude of cell proliferation forLibraries 3A and 3B for days 35 and 21, respectively, are provided inTables 12 and 13. Examples of first intracellular domains or portionsand/or mutants thereof, that were present in constructs in the repeatedscreens referred to as Libraries 3.1A and 3.1B that promoted cellproliferation are provided in Table 14 and 15.

For Library 3A, second intracellular (P4) domains from CD27, CD3G, CD40,and CD79B, or mutants thereof that are known to promote signalingactivity in certain cell types if such mutants are present in thetables, when found at the second intracellular domain (P4) of candidatechimeric polypeptides elements herein, promoted cell proliferation ofPBMCs between day 7 and day 21, when data is considered in a combinedmanner for all constructs with a second intracellular domain derivedfrom that gene (data not shown). This conclusion is based on enrichmentof sequence counts of constructs in mixed cultured PBMC cellpopulations, such that enrichment, calculated as the logarithm in base 2of the ratio between normalized count at day 21 plus one and normalizedcount at day 7 plus one, was at least 2 for Library 3A, when results forall constructs for a gene are combined. For Library 3B, secondintracellular domains from CD40, or mutants thereof that are known topromote signaling activity in certain cell types if such mutants arepresent in the tables, were the best performers when analyzed in thisway. Examples of second intracellular domains or portions and/or mutantsthereof that were present in constructs that promoted the largestmagnitude of cell proliferation for Libraries 3A and 3B for days 35 and21, respectively, are provided in Tables 12 and 13. Examples of secondintracellular domains or portions and/or mutants thereof, that werepresent in constructs in the repeated screens referred to as Libraries3.1A and 3.1B that promoted the largest magnitude of cell proliferationare provided in Table 14 and 15.

With respect to specific genes, of the 798 constructs with CD8B (CD8bmolecule) as the P2 part, 111 were positive, which was defined asconstructs having an enrichment above two (log 2) for Libraries 3A or 3Bon day 21. Of the 1,032 constructs with CD40 (CD40 molecule) as the P2part, 159 were positive. Of the 1,339 constructs with CRLF2 (cytokinereceptor like factor 2) as the P2 part, 175 were positive. Of the 593constructs with CSF2RA (colony stimulating factor 2 receptor alphasubunit) as the P2 part, 108 were positive. Of the 714 constructs withFCGR2C (Fc fragment of IgG receptor IIc (gene/pseudogene)) as the P2part, 88 were positive. Of the 788 constructs with ICOS (inducibleT-cell costimulator) as the P2 part, 108 were positive. Of the 882constructs with IFNAR1 (interferon alpha and beta receptor subunit 1) asthe P2 part, 106 were positive. Of the 806 constructs with IFNGR1(interferon gamma receptor 1) as the P2 part, 104 were positive. Of the938 constructs with IL3RA (interleukin 3 receptor subunit alpha) as theP2 part, 132 were positive. Of the 746 constructs with IL10RB(interleukin 10 receptor subunit beta) as the P2 part, 99 were positive.Of the 814 constructs with IL18R1 (interleukin 18 receptor 1) as the P2part, 131 were positive. Of the 552 constructs with IL18RAP (interleukin18 receptor accessory protein) as the P2 part, 83 were positive. Of the1,390 constructs with LEPR (leptin receptor) as the P2 part, 184 werepositive. Of the 795 constructs with PRLR (prolactin receptor) as the P2part, 123 were positive.

Of the 1,259 constructs with ILIRAP (interleukin 1 receptor accessoryprotein) as the P3 part, 158 were positive, which was defined asconstructs having an enrichment above two for Libraries 3A or 3B on day21. Of the 200 constructs with IL17RD (interleukin 17 receptor D) as theP3 part, 31 were positive. Of the 11 constructs with IL17RE (interleukin17 receptor E) as the P3 part, 3 were positive. Of the 538 constructswith IL23R (interleukin 23 receptor) as the P3 part, 86 were positive.Of the 1,531 constructs with MPL (MPL proto-oncogene, thrombopoietinreceptor) as the P3 part, 509 were positive.

Of the 3,124 constructs with CD3G (CD3g molecule) as the P4 part, 458were positive, which was defined as constructs having an enrichmentabove two for Libraries 3A or 3B on day 21. Of the 3,293 constructs withCD27 (CD27 molecule) as the P4 part, 443 were positive. Of the 5,163constructs with CD40 (CD40 molecule) as the P4 part, 724 were positive.Of the 4,486 constructs with CD79B (CD79b molecule) as the P4 part, 663were positive. It is noteworthy for any construct that since this is acompetitive assay, “negative” means that a construct was out competedwith respect to promoting proliferation, but not that a construct is notcapable of promoting proliferation.

For Libraries 3A and 3.1A, the constructs E008/E013-T041-S186-S050,E006/E011-T077-S186-S211, E007/E012-T021-S186-S051,E009/E014-T041-S186-S053, E007/E012-T073-S186-S053,E006/E011-T017-S186-S051, E006/E011-T031-S186-S211,E006/E011-T011-S186-S050, E006/E011-T011-S186-S047,E007/E012-T001-S186-S050, E006/E011-T041-S186-S051,E008/E013-T028-S186-S076, E009/E014-T029-S199-S053,E009/E014-T062-S186-S216, E007/E012-T006-S058-S051,E009/E014-T076-S186-S211, and E007/E012-T001-S186-S047 had particularlynoteworthy enrichments in both screens, where the P1 parts separated bya slash here included different tags for Libraries 3A and 3A.1 as shownin Tables 6, 12, and 14. For the purposes of the repeated screens,constructs with particularly noteworthy enrichments were those that hada log₂((normalized count data on the last day+1)/(normalized count dataon day 7+1)) value above 2.

Further information about the first and second intracellular domains inconstructs with particularly noteworthy enrichments in screens of bothLibrary 3A and Library 3.1A is provided in Table 21, including thegene(s) the first and second intracellular domain are derived from,whether the first and/or second intracellular domain are cytokinereceptors, and whether the first and/or second intracellular domain haveat least one ITAM motif. Constructs with intracellular domains from MPL,OSMR, or CSF2RA, when present at the first intracellular domain (P3),and constructs with intracellular domains from CD40, TNFRSF4, CD79B,CD27, FCGR2A, or TNFRSF18, when present at the second intracellulardomain (P4), showed particularly noteworthy enrichments in bothLibraries 3A and 3.1A (Table 21). Constructs with domains from thecytokine receptors MPL, OSMR, or CSF2RA, when present at the firstintracellular domain (P3), and constructs with domains from the cytokinereceptors CD40, TNFRSF4, CD27, FCGR2A, or TNFRSF18, when present at thesecond intracellular domain (P4), showed particularly noteworthyenrichments in both Libraries 3A and 3.1A (Table 21). Constructs withdomains containing ITAM motifs from MPL or OSMR, when present at thesecond intracellular domain (P4), showed particularly noteworthyenrichments in both Libraries 3A and 3.1A (Table 21).

For Libraries 3B and 3.1B, the constructs E007/E012-T017-S186-S051,E007/E012-T073-S186-5053, E008/E013-T028-S186-S047,E006/E011-T011-S186-S047, E007/E012-T082-S176-S214,E006/E011-T046-S186-S052, E008/E013-T029-S186-S052,E009/E014-T011-S186-S053, E008/E013-T032-S186-S039,E007/E012-T034-S186-S051, E007/E012-T041-S192-S213,E006/E011-T014-S069-5213, E006/E011-T022-S186-S053,E006/E011-T023-S115-S075, E006/E011-T029-S106-S213,E006/E011-T032-S155-S080, E006/E011-T041-S186-S216,E006/E011-T057-S135-S080, E006/E011-T072-S191-X002,E006/E011-T077-S186-S216, E006/E011-T080-S141-S080,E007/E012-T001-X001-5214, E007/E012-T007-S059-S211,E007/E012-T016-S186-S052, E007/E012-T031-S186-S053,E007/E012-T044-S102-S052, E007/E012-T044-S142-X002,E007/E012-T055-S069-S053, E007/E012-T063-S176-S216,E007/E012-T065-S157-S075, E008/E013-T008-S085-X002,E008/E013-T011-S085-5048, E008/E013-T021-S109-X002,E008/E013-T021-S168-S211, E008/E013-T032-S064-S214,E008/E013-T037-S170-S215, E008/E013-T038-S176-S048,E008/E013-T039-S137-S216, E008/E013-T041-S141-S053,E008/E013-T045-S177-S048, E008/E013-T048-S109-S074,E008/E013-T073-S199-5075, E009/E014-T001-S157-S074,E009/E014-T005-S196-S049, E009/E014-T011-S130-X002,E009/E014-T013-S155-X002, E009/E014-T017-S186-S076,E009/E014-T021-S142-S080, E009/E014-T023-S082-S076,E009/E014-T038-S196-S037, E009/E014-T055-S186-S052,E009/E014-T060-S175-5053, E009/E014-T070-S085-S212,E008/E013-T026-S054-S213, E009/E014-T007-S120-S053,E007/E012-T045-S186-S211, E008/E013-T073-S186-X002,E008/E013-T074-S186-X002, E007/E012-T055-S186-S053,E008/E013-T036-S186-S053, E007/E012-T017-S058-S053,E008/E013-T030-S189-5080, E006/E011-T029-S081-S047,E009/E014-T044-S194-S050, E006/E011-T028-S121-X002,E008/E013-T028-S186-S053, E009/E014-T078-S142-S213,E009/E014-T041-S186-S051, E008/E013-T006-S186-S050,E006/E011-T028-S186-S075, E006/E011-T040-S120-S038,E007/E012-T044-S115-S211, E009/E014-T039-S176-S075,E007/E012-T028-S186-S050, E008/E013-T031-S202-S050,E007/E012-T072-S192-S053, E006/E011-T065-X001-S051,E007/E012-T030-S062-X002, E007/E012-T073-S186-X002,E009/E014-T056-S186-S053, E008/E013-T046-S137-X002,E006/E011-T016-S136-5076, E007/E012-T032-S142-S037,E007/E012-T065-S120-S215, E009/E014-T077-S186-S047,E009/E014-T001-S126-S051, E006/E011-T030-S121-S039,E008/E013-T006-S176-S213, E009/E014-T032-S130-S215,E008/E013-T041-S186-S039, E009/E014-T021-S186-S047,E008/E013-T026-S137-S214, E007/E012-T029-S116-S075,E008/E013-T026-S106-S049, and E007/E012-T032-S168-S075 had particularlynoteworthy enrichments in both screens, where the P1 parts separated bya slash here included different tags for Libraries 3B and 3.1B as shownin Tables 6, 13, and 15. For the purposes of the repeated screens,constructs with particularly noteworthy enrichments were those that hada log 2₂((normalized count data on the last day+1)/(normalized countdata on day 7+1)) value above 2.

Further information about the first and second intracellular domains inconstructs with particularly noteworthy enrichments in screens of bothLibrary 3B and Library 3.1B is provided in Table 22, including thegene(s) the first and second intracellular domain are derived from,whether the first and/or second intracellular domain are cytokinereceptors, and whether the first and/or second intracellular domain haveat least one ITAM motif. Constructs with intracellular domains from MPL,LEPR, MYD88, EPOR, IL5RA, IL2RG, IL18RAP, IL11RA, IL13RA1, CSF2RA,IL1RL1, IL13RA2, IL20RB, IFNGR2, IL3RA, IL27RA, CSF3R, IL31RA, IL12RB1,OSMR, IL10RB, IFNAR2, CRLF2, IL7R, IFNAR1, PRLR, IL9R, IL6R, or IL15RA,when present at the first intracellular domain (P3), and constructs withintracellular domains from CD40, CD79B, CD27, TNFRSF14, CD79A, CD3G,TNFRSF9, FCGR2C, ICOS, TNFRSF18, TNFRSF4, CD28, FCER1G, FCGR2A, CD3D,TNFRSF8, or CD3E, when present at the second intracellular domain (P4),showed particularly noteworthy enrichments in both Libraries 3B and 3.1B(Table 22). Constructs with domains from the cytokine receptors MPL,LEPR, EPOR, IL5RA, IL2RG, IL18RAP, IL11RA, IL13RA1, CSF2RA, ILIRL1,IL13RA2, IL20RB, IFNGR2, IL3RA, IL27RA, CSF3R, IL31RA, IL12RB1, OSMR,IL10RB, IFNAR2, CRLF2, IL7R, IFNAR1, PRLR, IL9R, IL6R, or IL15RA, whenpresent at the first intracellular domain (P3), and constructs withdomains from the cytokine receptors CD40, CD27, TNFRSF14, TNFRSF9,FCGR2C, TNFRSF18, TNFRSF4, FCER1G, FCGR2A, or TNFRSF8, when present atthe second intracellular domain (P4), showed particularly noteworthyenrichments in both Libraries 3B and 3.1B (Table 22). Constructs withdomains containing ITAM motifs from CD79B, CD79A, CD3G, FCGR2C, FCER1G,FCGR2A, CD3D, or CD3E, when present at the second intracellular domain(P4), showed particularly noteworthy enrichments in both Libraries 3Band 3.1B (Table 22).

FACs analysis revealed that the Day 49 Library 3A expanded cells wereprimarily CD3+, CD8+, CD56+, and FLAG-Tag+. The table immediately belowshows the percentage of lymphocytes that stained positive for theindicated marker and marker combinations. This data shows that thedrivers identified from this library screen were primarily expanding theCD3+ T cell component.

% of Lymphocytes CD3+ 98.11% CD3+CD4+ 8.96% CD3+CD8+ 87.00% CD3-CD56+0.22% CD3+CD56+ 79.13% CD3+FLAG+ 79.93% CD3+CD4+FLAG+ 0.77%CD3+CD8+FLAG+ 79.66% CD3-CD56+FLAG+ 3.33% CD3+CD56+FLAG+ 78.14%CD19+FLAG+ 0.03%

For Library 4B, which included constructs that encoded the chimericpolypeptides but no CAR, and transduced PBMCs that were not supplementedwith fresh untransduced PBMCs, 154 top candidates were identified (SeeTable 16) that promoted the largest magnitude of PBMC proliferationbetween day 7 and the last day indicated on the table when cultured inthe absence of IL-2.

For Library 4B, transmembrane domains (P2) from CD40, CD8B, CSF2RA,IL18R1, and IL3RA, or mutants thereof that are known to promoteconstitutive signaling activity in certain cell types if such mutantsare present in the constructs provided in the tables, when found at thetransmembrane domain position (P2) of candidate chimeric polypeptideselements herein, promoted cell proliferation of PBMCs between day 7 andthe last day indicated on the table, when data is considered in acombined manner for all constructs with a transmembrane domain derivedfrom that gene. This conclusion is based on enrichment of sequencecounts of constructs in mixed cultured PBMC cell populations, such thatenrichment, calculated as the logarithm in base 2 of the ratio betweennormalized count at the last day indicated on the table plus one andnormalized count at day 7 plus one, was at least 2 for Library 4B, whenresults for all constructs for a gene are combined. Examples oftransmembrane domains or portions and/or mutants thereof, that werepresent in Library 4B in constructs that promoted cell proliferation areprovided in Tables 16. Examples of transmembrane domains or portionsand/or mutants thereof, that were present in constructs in the repeatedscreen referred to as Library 4.1B that promoted cell proliferation areprovided in Table 17.

For Library 4B, first intracellular (P3) domains from CRLF2, CSF2RA,IFNGR2, IL4R, MPL, and OSMR or mutants thereof that are known to promotesignaling activity in certain cell types if such mutants are present inthe constructs provided in the tables, when found at the firstintracellular domain (P3) of candidate chimeric polypeptides elementsherein, promoted cell proliferation of PBMCs between day 7 and the lastday indicated on the table, when data is considered in a combined mannerfor all constructs with a first intracellular domain derived from thatgene. This conclusion is based on enrichment of sequence counts ofconstructs in mixed cultured PBMC cell populations, such thatenrichment, calculated as the logarithm in base 2 of the ratio betweennormalized count at the last day indicated on the table plus one andnormalized count at day 7 plus one, was above 0 for Library 4B, whenresults for all constructs for a gene are combined. Examples of firstintracellular domains or portions and/or mutants thereof, that werepresent in constructs in Library 4B that promoted cell proliferation areprovided in Table 16. Examples of first intracellular domains orportions and/or mutants thereof, that were present in constructs in therepeated screen referred to as Library 4.1B that promoted cellproliferation are provided in Table 17.

For Library 4, second intracellular (P4) domains from CD27, CD40, andCD79B, or mutants thereof that are known to promote signaling activityin certain cell types if such mutants are present in the constructsprovided in the tables, when found at the second intracellular domain(P4) of candidate chimeric polypeptides elements herein, promotedproliferation of PBMCs between day 7 and the last day indicated on thetable, when data is considered in a combined manner for all constructswith a second intracellular domain derived from that gene. Thisconclusion is based on enrichment of sequence counts of constructs inmixed cultured PBMC cell populations, such that more counts for suchdomains were present at the last day than day 7 for Library 4B, whenresults for all constructs for a gene are combined. Examples of secondintracellular domains or portions and/or mutants thereof, that werepresent in constructs in Library 4B that promoted cell proliferation areprovided in Table 16. Examples of second intracellular domains orportions and/or mutants thereof, that were present in constructs in therepeated screen referred to as Library 4.1B that promoted cellproliferation are provided in Table 17.

For Libraries 4B and 4.1B, the constructs E007/E012-T078-S154-S047,E008/E013-T062-S186-X002, E008/E013-T055-S186-S050,E009/E014-T057-S186-S050, E007/E012-T077-S054-S053,E007/E012-T034-S135-S211, E009/E014-T071-X001-S216,E009/E014-T011-S141-S037, E008/E013-T041-S186-S037,E006/E011-T038-S106-S039, E006/E011-T011-S121-X002,E007/E012-T007-S085-S215, E006/E011-T041-S186-S050,E007/E012-T008-S064-S051, E006/E011-T041-S186-S047,E008/E013-T045-S186-S051, E008/E013-T003-S104-S216,E006/E011-T019-S186-S053, E008/E013-T071-S064-S080,E006/E011-T021-S054-X002, E006/E011-T003-S135-X002,E009/E014-T020-S199-S213, E008/E013-T027-S121-S211,E009/E014-T032-S195-X002, E009/E014-T050-S171-X002,E008/E013-T069-S083-X002, E008/E013-T026-S116-S038,E009/E014-T072-S195-X002, E007/E012-T047-S058-S211,E008/E013-T046-S142-S080, E006/E011-T065-S186-S076,E006/E011-T062-S069-X002, E007/E012-T047-S098-X002,E009/E014-T069-S099-S048, E008/E013-T039-S141-S050,E006/E011-T052-S130-S052, E008/E013-T041-S186-X002,E007/E012-T019-S120-X002, E008/E013-T045-S186-S053,E006/E011-T003-S170-S039, E007/E012-T047-S058-S051,E007/E012-T069-S109-X002, E009/E014-T019-S130-S075, andE006/E011-T047-S054-S053 had particularly noteworthy enrichments in bothscreens, where the P1 parts separated by a slash here included differenttags for Libraries 4B and 4.1B as shown in Tables 6, 16, and 17. For thepurposes of the repeated screens, constructs with particularlynoteworthy enrichments were those that had a log₂((normalized count dataon the last day+1)/(normalized count data on day 7+1)) value above 2.

Further information about the first and second intracellular domains inconstructs with particularly noteworthy enrichments in screens of bothLibrary 4B and Library 4.1B is provided in Table 23, including thegene(s) the first and second intracellular domain are derived from,whether the first and/or second intracellular domain are cytokinereceptors, and whether the first and/or second intracellular domain haveat least one ITAM motif. Constructs with intracellular domains fromIL18R1, MPL, CRLF2, IL11RA, IL13RA1, IL2RG, IL7R, IFNGR2, CSF3R, IL2RA,OSMR, MYD88, IL31RA, IFNAR2, IL6R, CSF2RA, IL13RA2, EPOR, IL1R1, IL10RB,or IL3RA, when present at the first intracellular domain (P3), andconstructs with intracellular domains from CD27, CD40, CD79B, TNFRSF4,TNFRSF18, CD3D, CD3G, CD27, ICOS, TNFRSF9, CD3E, FCGR2A, CD28, CD79A, orFCGR2C, when present at the second intracellular domain (P4), showedparticularly noteworthy enrichments in both Libraries 4B and 4.1B (Table23). Constructs with domains from the cytokine receptors IL18R1, MPL,CRLF2, IL11RA, IL13RA1, IL2RG, IL7R, IFNGR2, CSF3R, IL2RA, OSMR, IL31RA,IFNAR2, IL6R, CSF2RA, IL13RA2, EPOR, IL1R1, IL10RB, or IL3RA, whenpresent at the first intracellular domain (P3), and constructs withdomains from the cytokine receptors CD27, CD40, TNFRSF4, TNFRSF18,TNFRSF9, FCGR2A, or FCGR2C, when present at the second intracellulardomain (P4), showed particularly noteworthy enrichments in bothLibraries 4B and 4.1B (Table 23). Constructs with domains containingITAM motifs from CD79B, CD3D, CD3G, CD3E, FCGR2A, CD79A, or FCGR2C, whenpresent at the second intracellular domain (P4), showed particularlynoteworthy enrichments in both Libraries 4B and 4.1B (Table 23).

For Library 4B, of the 572 constructs with CD8B (CD8b molecule) as theP2 part, 21 were positive, which was defined as constructs having anenrichment above two for Library 4B. Of the 897 constructs with CD40(CD40 molecule) as the P2 part, 50 were positive. Of the 551 constructswith CSF2RA (colony stimulating factor 2 receptor alpha subunit) as theP2 part, 25 were positive. Of the 732 constructs with IL3RA (interleukin3 receptor subunit alpha) as the P2 part, 47 were positive. Of the 738constructs with IL18R1 (interleukin 18 receptor 1) as the P2 part, 44were positive. Of the 1,683 constructs with MPL (MPL proto-oncogene,thrombopoietin receptor) as the P3 part, 305 were positive.

In the initial interim analysis for Library 3A at day 21, 66 of the top100 hits and 538 of the top 1000 hits had been decoded. Further decodingprovided deep decoding and the identity of 99 of the top 100 hits and992 of the top 1000 hits of Library 3A and data was collected for day 35and analysis was updated at day 21. In the initial analysis for Library3B at day 21, 77 of the top 100 hits and 464 of the top 1000 hits hadbeen decoded in the initial interim analysis. Further decoding provideddeep decoding and the identity of 100 of the top 100 hits and 708 of thetop 1000 hits of Library 3B. The top constructs for Libraries 3A and 3Bwith day 35 data for Library 3A and day 21 data for Library 3B are shownin Tables 12 and 13, respectively.

In Libraries 3A and 3B, which included constructs that encoded thechimeric polypeptides and a CAR and transduced PBMCs that weresupplemented with (Library 3A) or without (Library 3B) freshuntransduced PBMCs, after deep decoding 124 top candidates wereidentified for Library 3A at day 35, and 131 top candidates wereidentified for library 3B at day 21 (See Tables 12 and 13, respectively)that promoted the largest magnitude of PBMC proliferation between day 7and the last day indicated on the table when cultured in the absence ofIL-2 (i.e. IL-2 was not added to the culture medium during culturingafter the initial transduction). The top candidate lists of Tables 12(Library 3A day 35) and 13 (Library 3B day 21) were generated bycombining the top 100 most prevalent candidates at day 21 (Library 3B)or day 21 or 35 (Library 3A) and the top 100 most enriched candidateconstructs between day 21 and day 7 (Library 3B) or between day 21 andday 7 or day 35 and day 7 (Library 3A) after deep decoding.

From the top hits tables using results with the deep decoding certainpolypeptides in specific modules of the CLEs were among the top hitsacross at least one of Libraries 3A and 3B and for at least one of thetime points of day 21 and day 35 (Library 3A) or day 21 (Library 3B).For example, exemplary constructs containing CD40 (specifically forexample the transmembrane domain with the code T011), ICOS (specificallyfor example the transmembrane domain with the code T028), FCGR2C(specifically for example transmembrane domain with the code T024), PRLR(specifically for example the transmembrane domain with the code T077),IL3RA (specifically for example the transmembrane domain with the codeT041), IL6ST (specifically for example the transmembrane domain with thecode T045) in the P2 position, IL18R1 (specifically for example thetransmembrane domain with the code T062) in the P2 position; MPL(specifically for example the intracellular domain with the code S186),IL18R1 (specifically for example the intracellular domain with the codeS154), IL13RA2 (specifically for example the intracellular domain withthe code S142), IL10RB (specifically for example the intracellulardomain with the code S130), LEPR (specifically for example intracellulardomains with the code S176), IFNAR2 (specifically for example theintracellular domain with the code S083), IL23R (specifically forexample the intracellular domain with the code S165), MyD88(specifically for example the intracellular domain with the code S194),and CSF2RA (specifically for example the intracellular domain with thecode S058), in the P3 position; and/or CD40 (specifically for example anintracellular domain with the code S050 or S051), CD79B (specificallyfor example the intracellular domain with the code S053), TNFRSF4specifically for example the intracellular domain with the code S211),TNFRSF9 (specifically for example the intracellular domain with the codeS213), TNFRSF14 (specifically for example the intracellular domain withthe code S214), FCGRA2 (specifically for example the intracellulardomains with the code S076), CD3G (specifically for example theintracellular domain with the code S039), or CD27 (specifically forexample the intracellular domain with the code S047) in the P4 positionwere among the top 5 most frequent chimeric polypeptides (see Tables 12and 13). Strikingly, MPL was the most frequent chimeric polypeptide atP3 by a large margin for both Libraries 3A and 3B. Note the top hitsparts from P3 were not included in the P4 position for these constructsin Library 3A or 3B.

The following additional observations are noteworthy: P2_CD40, P2_ICOS,P3_MPL, P4_CD40, and P4_CD79B came up as most frequent parts in the tophits in each of Tables 12 and 13. P4_CD27 came up as a most frequentpart in Tables 12 and 13. CSF2RB came up 4 times in top 123 mostenriched and most rep at P2, but mutant V449E mutant did not occur inthese top hits (Table 12). Accordingly, in some embodiments herein achimeric polypeptide comprises a transmembrane domain identified in anyof the constructs shown in Tables 12 and 13 other than a transmembranedomain mutant V449E of CSF2RB. Eight MyD88 variants occurred at the P3position at least once in the Library 3A top 123 (Table 12). Bothvariants of CD40 tested in the library occurred at least once at the P4position in the top 123 list for Library 3A (Table 12). The combinationof MPL at P3 and CD40 at P4 showed up in 26 constructs in the list ofthe top 123 constructs of Library 3A (Table 12).

For gene by gene analysis, for Library 3A, transmembrane domains (P2)from CD4, CD8B, CD40, CRLF2, CSF2RA, CSF3R, EPOR, FCGR2C, GHR, ICOS,IFNAR1, IFNGR1, IFNGR2, IL1R1, ILIRAP, IL2RG, IL3RA, IL5RA, IL6ST,IL7RA, IL10RB, IL11RA, IL13RA2, IL17RA, IL17RB, IL17RC, IL17RE, IL18R1,IL18RAP, IL20RA, IL22RA1, IL31RA, LEPR, PRLR, and TNFRSF8, or mutantsthereof that are known to promote signaling activity in certain celltypes if such mutants are present in the constructs provided in thetables, promoted proliferation of PBMCs between day 7 and the last dayindicated on the table, when data is considered in a combined manner forall constructs with a transmembrane domain derived from that gene (Table12). This conclusion is based on enrichment of sequence counts ofconstructs in mixed cultured PBMC cell populations, such thatenrichment, calculated as the logarithm in base 2 of the ratio betweennormalized count at the last day indicated on the table plus one andnormalized count at day 7 plus one, was at least 2 for Library 3A, whenresults for all constructs for a gene are combined. For Library 3B(Table 13), transmembrane domains from CD40, ICOS, and IL18R1, ormutants thereof that are known to promote signaling activity in certaincell types if such mutants are present in the constructs provided in thetables, were the best performers when analyzed in this way. Examples oftransmembrane domains or portions and/or mutants thereof, that werepresent in constructs that promoted the largest magnitude of cellproliferation for Library 3A and 3B are provided in Tables 12 and 13.Examples of transmembrane domains or portions and/or mutants thereof,that were present in constructs in the repeated screens referred to asLibraries 3.1A and 3.1B that promoted cell proliferation are provided inTable 14 and 15.

For Library 3A, first intracellular (P3) domains from CSF2RA, IFNAR1,ILIRAP, IL4R, IL6ST, IL11RA, IL12RB2, IL17RA, IL17RD, IL17RE, IL18R1,IL21R, IL23R, MPL, and MyD88, or mutants thereof that are known topromote signaling activity in certain cell types if such mutants arepresent in the constructs provided in the tables, when found at thefirst intracellular domain (P3) of candidate chimeric polypeptideselements herein, promoted cell proliferation of PBMCs between day 7 andthe last day indicated on the table, when data is considered in acombined manner for all constructs with a first intracellular domainderived from that gene. This conclusion is based on enrichment ofsequence counts of constructs in mixed cultured PBMC cell populations,such that enrichment, calculated as the logarithm in base 2 of the ratiobetween normalized count at the last day indicated on the table plus oneand normalized count at day 7 plus one, was at least 2 for Library 3A,when results for all constructs for a gene are combined. For Library 3B,first intracellular domains from CSF2RB, IL4R, and MPL, or mutantsthereof that are known to promote signaling activity in certain celltypes if such mutants are present in the constructs provided in thetables, were the best performers when analyzed in this way. Examples offirst intracellular domains or portions and/or mutants thereof that werepresent in constructs that promoted the largest magnitude of cellproliferation for Libraries 3A and 3B are provided in Tables 12 and 13.Examples of first intracellular domains or portions and/or mutantsthereof, that were present in constructs in the repeated screensreferred to as Libraries 3.1A and 3.1B that promoted cell proliferationare provided in Table 14 and 15.

For Library 3A, second intracellular (P4) domains from CD3D, CD3G, CD27,CD40, CD79A, CD79B, FCER1G, FCGRA2, ICOS, TNFRSF4, and TNFRSF8, ormutants thereof that are known to promote signaling activity in certaincell types if such mutants are present in the constructs provided in thetables, when found at the second intracellular domain (P4) of candidatechimeric polypeptides elements herein, promoted proliferation of PBMCsbetween day 7 and day 21 or between day 7 and the last day indicated onthe table, when data is considered in a combined manner for allconstructs with a second intracellular domain derived from that gene.This conclusion is based on enrichment of sequence counts of constructsin mixed cultured PBMC cell populations, such that enrichment,calculated as the logarithm in base 2 of the ratio between normalizedcount at the last day indicated on the table plus one and normalizedcount at day 7 plus one, was at least 2 for Library 3A, when results forall constructs for a gene are combined. For Library 3B, secondintracellular domains from CD40, or mutants thereof that are known topromote signaling activity in certain cell types if such mutants arepresent in the tables, although not achieving a 2-fold log 2 enrichment,were the best performers when analyzed in this way. Examples of secondintracellular domains or portions and/or mutants thereof that werepresent in constructs that promoted the largest magnitude of cellproliferation for Libraries 3A and 3B are provided in Tables 12 and 13.Examples of first intracellular domains or portions and/or mutantsthereof, that were present in constructs in the repeated screensreferred to as Libraries 3.1A and 3.1B that promoted cell proliferationare provided in Table 14 and 15.

Example 19. Thermal Denaturation of F1A-795 in the Absence and Presenceof Acyclovir by Differential Scanning Calorimetry (DSC)

In this example, the binding of a nucleoside analogue antiviral drug toan aptamer domain of a riboswitch (SEQ ID NO:87) was demonstrated bycomparing the thermal denaturation of the aptamer domain in the absenceversus presence of the nucleoside analogue antiviral drug acyclovir.

Aptamer Preparation

The T7 primer (SEQ ID NO:246) as well as the template (reversecomplement) of the identified candidate sequence was synthesized by IDT(Coralville, IA) as single-stranded DNA. Candidates were primer-extendedby combining components to 1 μM template, 2 μM T7 primer, 200 μM dNTPs,1× Titanium Taq DNA Polymerase, and 1× Titanium Taq buffer (ClontechLaboratories; Mountain View, CA), then heating for 3 minutes at 95° C.,1 minute at 55° C., and 2 minutes at 68° C. (no cycling). 42 μmoles ofdouble-stranded DNA were used for twelve 20-1 reactions with theAmpliscribe T7 High Yield Transcription Kit (Lucigen; Middleton, WI)according to standard kit directions (1× reaction buffer, 7.5 mM eachNTP, 10 mM DTT, 1× RiboGuard RNase inhibitor, 1× Ampliscribe T7 enzymesolution). Reactions were incubated at 42° C. overnight, then stopped byadding 1 μl of DNase I and incubating for 15 minutes at 37° C.Transcription products were purified on 10% denaturing PAGE with 8 Murea. At least 10 nmole of each candidate was lyophilized, andresuspended in 1× Binding Buffer (50 mM HEPES, 100 mM KCl, 0.5 mM MgCl₂,pH 7.3) on the day of binding assessment.

Briefly, DSC was used to analyze F1A-795 (7.2 μM) in the absence ofacyclovir or F1A-795 (2.77 μM) in the presence of acyclovir (29.4 μM).All analyses were conducted in 1× Binding Buffer and all water used wasDEPC-treated. All analytes were resuspended first in DEPC-treated waterthen diluted to their final listed concentration in 1× Binding Buffer.Prior to loading of the DSC (GE Healthcare MicroCal VP-DSC), the sampleswere degassed at 25° C. for 10 minutes. The sample without acyclovir wasscanned from 10° C. to 115° C. at a rate of 1° C. per minute. The samplewith acyclovir was scanned from 10° C. to 105° C. at a rate of 1° C. perminute.

Results

The thermal denaturation of F1A-795 in the absence of acyclovir asmeasured by DSC has a transition centered at about 60° C. (FIG. 28 ).This transition suggests the aptamer domain is structured in the absenceof acyclovir. In the presence of acyclovir, F1A-795 has a transitioncentered at about 75° C. (FIG. 28 ). This stabilizing effect indicatesthe nucleoside analogue antiviral drug acyclovir binds to the aptamerdomain F1A-795. Thus, this experiment confirmed that F1A-795 is bound byacyclovir.

Example 20. Transduction Efficiency of Freshly Isolated and UnstimulatedHuman T Cells by Retroviral Particles Containing a VSV-G PseudotypingElement, and a Membrane-Bound Activation Element, and OptionallyContaining Vpu Protein

Recombinant lentiviral particles were produced by transient transfectionof 293T cells (Lenti-X™ 293T, Clontech) with separate lentiviralpackaging plasmids encoding gag/pol and rev, and a pseudotyping plasmidencoding VSV-G. A third generation lentiviral expression vector encodingGFP, an anti-CD19 chimeric antigen receptor, and an eTAG referred toherein as F1-0-03 (FIG. 20 ) was co-transfected with the packagingplasmids. The cells were adapted to suspension culture by serial growthin Freestyle™ 293 Expression Medium (ThermoFisher Scientific). The cellsin suspension were seeded at 1×10⁶ cells/mL (30 mL) in a 125 mLErlenmeyer flask, and immediately transfected using polyethylenimine(PEI) (Polysciences) dissolved in weak acid.

Plasmid DNA was diluted in 1.5 ml Gibco™ Opti-MEM™ media for 30 mL ofcells. To obtain lentiviral particles pseudotyped with VSV-G, the totalDNA (1 μg/mL of culture volume) used was a mixture of 4 plasmids withthe following molar ratios: 2× genomic plasmid (F1-0-03), 1×Rev-containing plasmid, 1× VSV-G-containing plasmid, and 1×gag/pol-containing plasmid. To obtain lentiviral particles pseudotypedwith VSV-G and displaying anti-CD3-scFvFc-GPI on their surfaces, thetotal DNA (1 μg/mL of culture volume) used was a mixture of 5 plasmidswith the following molar ratios: 2× genomic plasmid (F1-0-03), 1×Rev-containing plasmid, 1× VSV-G-containing plasmid, 1×anti-CD3-scFvFc-GPI-containing plasmid, and 1× gag/pol-containingplasmid. To obtain lentiviral particles pseudotyped with VSV-G,displaying anti-CD3-scFvFc-GPI, and containing the accessory proteinVpu, the total DNA (1 μg/mL of culture volume) used was a mixture of 6plasmids with the following molar ratios: 2× genomic plasmid (F1-0-03),1× Rev-containing plasmid, 1× VSV-G-containing plasmid, 1×anti-CD3-scFvFc-GPI containing plasmid, 1× Vpu-CH-containing plasmid,and 1× gag/pol-containing plasmid. Separately, the PEI was diluted in1.5 ml Gibco™ Opti-MEM™ to 2 μg/mL (culture volume, 2:1 ratio to DNA).After a 5-minute room temperature incubation, the two solutions weremixed together thoroughly, and incubated at room temperature for 20 moreminutes. The final volume (3 ml) was added to the cells. The cells werethen incubated at 37° C. for 72 hours with rotation at 125 rpm and with8% CO₂. The antiCD3-scFvFc-GPI containing plasmid includes an scFvderived from UCHT1, and a GPI anchor attachment sequence derived fromDAF (CD55). The UCHT1scFvFc-GPI vector encodes a peptide (SEQ ID NO:278)that includes human Ig Kappa signal peptide (amino acids 1-22 of NCBI GINo. CAA45494.1) fused to the UCHT1 scFv (amino acids 21-264 of NCBI GINo. CAH69219.1), fused to human IgG1 Fc (amino acids 1-231 of NCBI GINo. AEV43323.1), fused to the human DAF GPI anchor attachment sequence(amino acids 345-381 of NCBI GI No. NP_000565). The Vpu-CH containingplasmid encodes a peptide(MFDFIARVDYRVGVVALVVALIIAIIVWSIVYIEYRKLLKQRKIDWLIKRIRERAEDSGNESDGEIEELSTMVDMEHLRLLDDL*) that includes the Vpu sequence from the HIV-1 MCH167-CC isolate (amino acids 1-84, GenBank: AGF30946.1).

After 72 hours, the supernatants were harvested and clarified bycentrifugation at 1,200 g for 10 minutes. The clarified supernatantswere decanted to a new tube. The lentiviral particles were precipitatedby overnight centrifugation at 3,300 g, at 4° C. The supernatant wasdiscarded, and the lentiviral particle pellets were resuspended in 1:100of initial volume of X-Vivo™ 15 medium (Lonza). Lentiviral particleswere titered by serial dilution and analysis of GFP expression, in 293Tand Jurkat cells, 72 hours post-transduction, by flow cytometry.

Peripheral blood mononuclear cells (PBMCs) were isolated from a buffycoat, collected and distributed by the San Diego Blood Bank, CA.SepMate™ 50 (Stemcell™)_based gradient density separation of PBMCs onFicoll-Paque PLUS® (GE Healthcare Life Sciences) was performed permanufacturers' instructions. 30 mL of buffy-coat diluted in X-Vivo™ 15medium were layered per each SepMate™ tube. After centrifugation at roomtemperature, at 1,200 g, for 20 min, the PBMC layers were collected,pooled and washed three times with 45 mL of X-Vivo™ 15 medium andcentrifugation at 400 g for 10 min at room temperature. The pellets werethen incubated at room temperature for 10 min in 10 mL of RBC lysisbuffer (Alfa Aesar) and washed an additional two times with 45 mL ofX-Vivo™ 15 medium, and centrifugation at 400 g for 10 min at roomtemperature. No additional steps were taken to remove monocytes. Afterisolation, fresh and unstimulated PBMCs were resuspended to a finalconcentration of 1×10⁶/mL in X-Vivo™ 15 medium, and were transduced, induplicates, with the lentiviral particles disclosed previously. Thetransductions were conducted for 3 days, at 37° C., 5% CO₂, in X-Vivo™15 medium. Transductions were conducted at MOI 10 for [VSV-g]pp and MOI1 for [VSV-G+UCHT1]pp and [VSV-G+UCHT1+Vpu-CH]pp, in a 12 wells plateformat, 1 mL/well, in duplicates. After 3 days of transduction, sampleswere collected and analyzed by FACS for absolute cell count and GFPexpression levels in the CD3+ population.

FIG. 34A and FIG. 34B show histograms of the percentage (%) CD3+ GFP+cells in the total CD3+ population (FIG. 34A) and a histogram of theabsolute cell count per well of the Live CD3+ population (FIG. 34B),respectively, 3 days after transduction of freshly isolated andunstimulated PBMCs with the indicated lentiviral particles. Each barrepresents the mean+/−SD of duplicates. FIG. 34A shows that adding anantiCD3-scFvFc-GPI moiety (UCHT1) to VSV-G pseudotyped lentiviralparticles results in higher transduction efficiencies of freshlyisolated and unstimulated PBMCs compared to [VSV-G]pp alone. FIG. 34Aalso shows that the addition of Vpu-CH can further improve thetransduction efficiency of the dually pseudotyped vector[VSV-G+UCHT1]pp. FIG. 34B shows that the UCHT1 moiety enhances cellstimulation by absolute cell count per uL as compared to compared toVSV-G only. The addition of Vpu and UCHT1 similarly enhances cellstimulation by absolute cell count as compared to VSV-G only.

Example 21. Characterization of Individual Lymphoproliferative Elements

In this example, select chimeric lymphoproliferative elements (CLEs)identified in the library screens described in Example 17 and Example 18were assessed individually. To further analyze CLEs identified in thescreens of Library 2B, activated PBMCs were transduced overnight withlentiviral particles encoding a CLE alone and cultured in the absence ofexogenous cytokines. To further analyze CLEs identified in the screensof Library 3A, activated PBMCs were transduced overnight with lentiviralparticles encoding a CLE flanked with an anti-CD19 CAR at the 5′ end,and cultured in the presence of donor-matched CD19+ B cells added to theculture every 7 days, but in the absence of exogenous cytokines. Thecells were cultured for up to 35 days to assess the ability of the CLEsto promote PBMC proliferation. This example further provides methods bywhich to characterize any individual putative lymphoproliferativeelement and further confirms the identities of several highly activeCLEs.

Recombinant lentiviral particles were produced in 293T cells (Lenti-X™293T, Clontech) that were adapted to suspension culture in Freestyle™293 Expression Medium (Thermo Fisher Scientific) serum-free chemicallydefined medium. The cells were transiently transfected using PEI with agenomic plasmid and 3 separate lentiviral packaging plasmids encodinggag/pol, rev, and a pseudotyping plasmid encoding VSV-G as explained inExample 20. Selected CLEs identified in the library screens described inExamples 17 and 18 were regenerated individually from their parts (P1-2,P3, and P4 or P1, P2, P3, and P4) and inserted into the same transgeneexpression cassettes as that of the library from which they were firstidentified. The module parts of each of the selected CLEs are shown inFIGS. 35 and 36 and the gene names and amino acids sequences are shownin Table 6. FIGS. 31 and 32 show the cassettes for Libraries 2 and 3,respectively. Cells transduced with lentiviral particles containingconstructs identified through library screens are referred to as “DL”followed by the library number. For example, cells transduced withlentiviral particles containing constructs from Library 2 are referredto as DL2. Thus, CLEs with the prefix “DL2” and “DL3” were constructedin the cassette shown in FIGS. 31 and 32 , respectively. Similarly,“DC1-5” and “DC1-6” comprise CLEs inserted into the transgene expressioncassette used for Library 1 as shown in FIG. 30 . The chimericlymphoproliferative element of DC1-5 encoded from amino to carboxyterminus, human IL-7 (SEQ ID NO:511), a flexible linker (SEQ ID NO:53),and residues 21-458 of the human IL-7Rα (SEQ ID NO:229) which is thefull length IL-7Rα without its signal peptide. The CLE of DC1-6 encodedfrom amino to carboxy terminus, IL-7 (SEQ ID NO:511), a flexible linker(SEQ ID NO:53), the extracellular and transmembrane portions of theIL-7Rα (CD127) (SEQ ID NO:513), and the intracellular domain of theIL-2RP (CD122) (SEQ ID NO:514). The chimeric proteins IL-7-IL-7Rα andIL-7-IL-7Rα-IL-2RP have been described previously (Hunter et al.Molecular Immunology 56(2013):1-11). The IL-7 “DC2-5” and “DC2-6”comprise CLEs inserted into the transgene expression cassette used forLibrary 2 as shown in FIG. 31 . The CLEs in DC2-5 and DC2-6 were thesame CLEs as DC1-5 and DC1-6, respectively. Untransduced PBMCs (NT),PMBCs transduced with a vector that encoded an eTag but not a CLE(F1-0-01), and PMBCs transduced with a vector that encoded an anti-CD19CAR and an eTag but not a CLE (F1-3-23) were included as negativecontrols.

The constructs from Library 2B used in this Example were: DL2-1(M024-S190-S047), DL2-2 (M025-S050-S197), DL2-3 (M036-S170-S047), DL2-4(M012-S045-S048), DL2-5 (M049-S194-S064), DL2-6 (M025-S190-S050), andDL2-7 (M025-S190-S051).

The constructs from Library 3A used in this Example were: DL3A-1(E013-T047-S158-S080), DL3A-2 (E011-T024-S194-S039), DL3A-3(E014-T040-S135-S076), DL3A-4 (E013-T041-S186-S051), DL3A-5(E013-T064-S058-S212), DL3A-6 (E013-T028-S186-S051), DL3A-7(E014-T015-S186-S051), DL3A-8 (E011-T016-S186-S050), DL3A-9(E011-T073-S186-S050), and DL3A-10 (E013-T011-S186-S211).

On Day 0, PBMCs were enriched from buffy coats (San Diego Blood Bank) bydensity gradient centrifugation with Ficoll-Paque PREMIUM® (GEHealthcare Life Sciences) according to the manufacturer's instructionsfollowed by lysis of red blood cells. 1.5×10⁶ viable PBMCs were seededin the wells of G-Rex 6 Well Plates (Wilson Wolf, 80240M) in 3 mlComplete OpTmizer™ CTS™ T-Cell Expansion SFM supplemented with 100 IU/ml(IL-2), and 50 ng/ml anti-CD3 antibody (317326, Biolegend) to activatethe PBMCs for viral transduction. After incubation overnight at 37° C.and 5% CO₂, lentiviral particles encoding the constructs described abovewere added directly to the activated PBMCs at an MOI of 5 and incubatedovernight at 37° C. and 5% CO₂. The following day, the media volume ineach well was brought to 30 ml with Complete OpTmizer™ CTS™ T-CellExpansion SFM and the plates were returned to the incubator. No IL-2,IL-7, or other exogenous cytokine was added at this or subsequent cellculture steps. The cells from each well were collected on Day 7 todetermine cell numbers, percent viability, and percent transduced cells,which was defined as the percent of FLAG-Tag+ or E-Tag+ cells by FACSanalysis. The cells were then centrifuged, resuspended in fresh CompleteOpTmizer™ CTS™ T-Cell Expansion SFM, and 0.5×10⁶ cells for each samplewere re-seeded into the well of a G-Rex 6 Well Plate in 30 ml ofComplete OpTmizer™ CTS™ T-Cell Expansion SFM. Previously cryopreservedDay 0 donor matched PBMCs containing 0.5×10⁶ CD19+ B cells (asdetermined by FACS on Day 0) were added to cultures of “DL3A-” toprovide CD19+ B-cell activation of the CD19 ASTR. This process wasrepeated on days 14, 20, and 28 before the cells were harvested on Day35.

Results

PBMCs transduced with lentiviral particles encoding CLEs were culturedin the absence of exogenous cytokines for 35 days. Proliferation isrepresented as fold expansion which was calculated by dividing the totalnumber of cells by the number of cells seeded for that time point. PBMCstransduced with each individual selected CLE shown in FIG. 35proliferated more than the negative controls, which includeduntransduced PBMCs and PMBCs transduced with a vector that encoded aneTag but not a CLE (FIG. 35 ). Furthermore, the following CLEs exhibiteda fold expansion greater than 23 at days 14, 21, 28, and 35 while thefold expansion of the negative controls was near 0 for these timepoints:DL2-7, DC2-6, DL2-6, DL2-2, DL2-1, and DC2-5. PBMCs transduced withconstructs encoding an anti-CD19 CAR and various CLEs (shown in FIG. 36) and cultured in the presence of fresh donor matched PBMCs added every7 days, but in the absence of exogenous cytokines, proliferated morethan the negative controls, which included untransduced PBMCs and PMBCstransduced with a vector that encoded an anti-CD19 CAR and an eTag butnot a CLE. All of the CLEs shown in FIG. 36 exhibited a fold expansiongreater than 23 at day 35 while the fold expansion of the negativecontrols was near 0 at day 35. The proliferation of PBMCs transducedwith lentiviral particles containing polynucleotides encoding DL3A-1(E013-T047-S158-S080), DL3A-2 (E011-T024-S194-S039), DL3A-3(E014-T040-S135-S076), and DL3A-5 (E013-T064-S058-S212) were comparableto the negative controls and are not shown on FIG. 36 .

Example 22. Proof of Concept for Various Illustrative Methods ProvidedHerein, Including In Vivo Expansion of Genetically Modified Lymphocytes

This example provides exemplary methods for transducing PBMCs, includingT cells, ex vivo and expanding those transduced PBMCs, including Tcells, in vivo. Such methods include an illustrative 4 hour transductionmethod with illustrative recombinant lentiviral vectors that expresscertain illustrative chimeric lymphoproliferative elements. Furthermore,such methods provide additional exemplary methods for transducing PBMCs,which in illustrative embodiments are typically T cells, withreplication incompetent recombinant retroviral particles in 4 hourswherein the replication incompetent recombinant retroviral particleswere produced by transfecting packaging cells with vectors encodingvarious components of the replication incompetent recombinant retroviralparticles in suspension in serum-free, chemically defined media.

Materials and Methods

Recombinant lentiviral particles were produced in 293T cells (Lenti-X™293T, Clontech) that were adapted to suspension culture in Freestyle™293 Expression Medium (Thermo Fisher Scientific) serum-free chemicallydefined medium. The cells were transiently transfected using PEI with 1of 3 genomic plasmids (detailed below) and 3 separate lentiviralpackaging plasmids encoding gag/pol, rev, and a pseudotyping plasmidencoding VSV-G as explained in Example 20. To produce retroviralparticles that also display a membrane-bound activation element, afourth packing plasmid encoding a membrane-bound polypeptide capable ofbinding to CD3 (UCHT1scFvFc-GPI) was co-transfected as described inExample 20. The genomic plasmids were third generation lentiviralexpression vectors containing a deletion in the 3′LTR leading toself-inactivation wherein the plasmids encoded the following:

-   -   (1) Anti-ROR2 MRB-CAR:T2A:eTag (F1-1-27): This genomic plasmid        is identical to that shown in FIG. 20 except that the sequence        encoding anti-CD19 CAR was replaced with an MRB-ASTR that has an        scFv that recognizes human ROR2, a CD8 stalk and transmembrane        sequence (SEQ ID NO:75), CD137 (SEQ ID NO:1), and CD3z (SEQ ID        NO:13). Recombinant lentiviral particles encoding this construct        are called F1-1-27;    -   (2) Flag-Anti-ROR2 MRB-CAR:T2A:CLE (F1-1-228 and F1-1-228U):        This genomic plasmid is identical to that shown in FIG. 20        except that the sequence encoding anti-CD19 was replaced with a        Flag-tag covalently linked to the anti-ROR2 MRB-CAR described        in (1) above, and the GMCSFR ss:eTag was replaced with a CLE.        The CLE was a type I transmembrane protein comprising an        extracellular dimerization module (P1), a transmembrane module        (P2), and 2 intracellular modules (P3 and P4). The genes in        positions P1-P2-P3-P4 were MycTag 2A Jun-IL13RA-MPL-CD40. The        codes for these modules are E013-T041-S186-S051 (See Table 6).        This CLE was first identified in libraries 3A, 3B, and 4B and        was the 6^(th) most enriched CLE between days 7 and 35 in        library 3A. Recombinant lentiviral particles encoding this        construct are called F1-1-228 and recombinant lentiviral        particles encoding this construct and displaying UCHT1scFvFc-GPI        are called F1-1-228U; or    -   (3) Flag-Anti-CD19 CAR:T2A:CLE (F1-3-219 and F1-3-219U): This        genomic plasmid is identical to the anti-human CD19 CAR shown in        FIG. 20 except that a Flag-tag was inserted between the CD8ss        and the CAR, and the sequence encoding the GMCSFRss:eTag was        replaced with a CLE. The CLE was a type I transmembrane protein        comprising an interleukin polypeptide covalently linked via a        flexible linker to the extracellular and transmembrane domains        of its cognate interleukin receptor and covalently linked to the        intracellular domain of a dissimilar cytokine receptor. In        particular, from amino to carboxy terminus the plasmid encoded        IL-7 (SEQ ID NO:511), a flexible linker (SEQ ID NO:53), the        extracellular and transmembrane portions of the IL-7Rα (CD127)        (SEQ ID NO:513), and the intracellular domain of the IL-2RP        (CD122) (SEQ ID NO: 514). Recombinant lentiviral particles        encoding this construct are called F1-3-219 and recombinant        lentiviral particles encoding this construct and displaying        UCHT1scFvFc-GPI are called F1-3-219U.        PBMC Isolation, Overnight Transduction of PBMCs after Ex Vivo        Stimulation, Followed by 15 Day Ex Vivo Expansion of Engineered        Lymphocytes

On Day 0, PBMCs were isolated from ACD peripheral blood from a healthyvolunteer with informed consent by density gradient centrifugation withFicoll-Pacque™ (General Electric) using a CS-900.2 kit (BioSafe; 1008)on a Sepax 2 S-100 device (Biosafe; 14000) according to themanufacturer's instructions. 3.0×10⁷ viable PBMCs were seeded in a 1 LG-Rex (Wilson-Wolf) and the volume was brought to 60 ml with CompleteOpTmizer™ CTS™ T-Cell Expansion SFM supplemented with 100 IU/ml IL-2(Novoprotein, GMP-CD66), 10 ng/ml IL-7 (Novoprotein, GMP-CD47), and 50ng/ml anti-CD3 antibody (OKT3, Novoprotein) to activate the PBMCs, whichincluded T cells and NK cells, for viral transduction. After incubationovernight at 37° C. and 5% CO₂, lentiviral particles encoding theanti-ROR2 MRB-CAR, F1-1-27, were added directly to the activated PBMCsat an MOI of 5 (440 ul) and incubated overnight at 37° C. and 5% CO₂.Following the overnight incubation, the cells were fed by bringing thetotal volume of media in the G-Rex to 100 ml with Complete OpTmizer™CTS™ T-Cell Expansion SFM supplemented with NAC (Sigma) to increase thefinal concentration by 10 mM along with 100 IU/ml recombinant human IL-2and 10 ng/ml recombinant human IL-7. The G-Rex device was incubated in astandard humidified tissue culture incubator at 37° C. and 5% CO₂ withadditions of 100 IU/ml recombinant human IL-2 and 10 ng/ml recombinanthuman IL-7 solution every 48 hours. The cells were expanded through Day15 before being harvested. These transduced cells were washed infreezing media (70% RPMI 1640, 20% heat-inactivated FBS, 10% DMSO) andcryopreserved in 1 ml aliquots at 5.0×10⁷ cells/ml for later use. Twodays prior to use in Group A of the experiments in this example below, 8vials (4.0×10⁸) of these cryopreserved F1-1-27 transduced PBMCs werethawed and rested for 2 days in a G-Rex100M containing 377 ml CompleteOpTmizer™ CTS™ T-Cell Expansion SFM (OpTmizer™ CTS™ T-Cell ExpansionBasal Medium 1 L (Thermo Fisher, A10221) supplemented with ml OpTmizer™CTS™ T-Cell Expansion Supplement (Thermo Fisher, A10484-02), CTS™ ImmuneCell SR (Thermo Fisher, A2596101), and CTS™ GlutaMAX™-I Supplement(Thermo Fisher, A1286001) according to manufacturer's instructions)supplemented with 100 IU/ml of IL-2 (Novoprotein), 10 ng/ml IL-7(Novoprotein), and sufficient NAC to increase the final concentration by10 mM.

PBMC Isolation and Advantageously Fast Transduction of RestingLymphocytes without Prior Ex Vivo Stimulation and without Ex Vivo CellExpansion

Whole human blood from 2 healthy volunteers with informed consent wascollected into multiple 100 mm Vacutainer tubes (Becton Dickenson;364606) containing 1.5 ml of Acid Citrate Dextrose Solution Aanticoagulant (ACD peripheral blood). For each volunteer, blood from theVacutainer tubes was pooled (185.2 ml for Group B, 182.5 ml for Group C)and distributed to 2 standard 500 ml blood collection bags. Thefollowing steps of PBMCs enrichment through transduction were performedin a closed system.

The blood in the 2 blood bags from each volunteer was processedsequentially using density gradient centrifugation with Ficoll-Paque™(General Electric) using a CS-900.2 kit (BioSafe; 1008) on a Sepax 2S-100 device (Biosafe; 14000) using 2 wash cycles according to themanufacturer's instructions, to obtain 45 ml of isolated PBMCs from eachrun. The wash solution used in the Sepax 2 process was Normal Saline(Chenixin Pharm)+2% human serum albumin (HSA) (Sichuan Yuanda ShuyangPharmaceutical). The final cell resuspension solution was 45 ml CompleteOpTmizer™ CTS™ T-Cell Expansion SFM (OpTmizer™ CTS™ T-Cell ExpansionBasal Medium 1 L (Thermo Fisher, A10221-03) supplemented with 26 mlOpTmizer™ CTS™ T-Cell Expansion Supplement (Thermo Fisher, A10484-02),25 ml CTS™ Immune Cell SR (Thermo Fisher, A2596101), and 10 ml CTS™GlutaMAX™-I Supplement (Thermo Fisher, A1286001)). Each processing stepon the Sepax 2 machine was approximately 1 hour and 12 minutes. EnrichedPBMCs obtained from the 2 processing runs were pooled separately forGroup B and Group C, and the cells counted.

5.5×10⁷ freshly enriched, viable PBMCs were seeded into each of 4standard blood collection bags for Group B and the volume was brought to55 ml with Complete OpTmizer™ CTS™ T-Cell Expansion SFM such that thecells were at a density of 1.0×10⁶/ml. 1.12×10⁸ freshly enriched, viablePBMCs were seeded into each of 2 standard blood collection bags forGroup C and the volume was brought to 110 ml with Complete OpTmizer™CTS™ T-Cell Expansion SFM such that the cells were at a density of1.0×10⁶/ml. No anti-CD3, anti-CD28, IL-2, IL-7, or other exogenouscytokine was added to activate or otherwise stimulate the lymphocyte exvivo prior to transduction. Lentiviral particles were added directly tothe non-stimulated PBMCs in blood collection bags at an MOI of 1 asfollows: 0.779 ml of F1-1-228 was added to one bag and 3.11 ml ofF1-1-228U was added to the other bag of Group B PBMCs; 0.362 ml ofF1-3-219 was added to one bag and 3.52 ml of F1-3-219U was added to theother bag of Group C PBMCs. The transduction reaction mixtures weregently massaged to mix the contents then incubated for four (4) hours inthe blood collection bags in a standard humidified tissue cultureincubator at 37° C. and 5% CO₂. The PBMCs from each bag were thentransferred to a 50 ml Conical tube (thus removing the cells from theclosed system in this proof of concept experiment) and washed 3 times inDPBS+2% HSA before being resuspended in 5 mls DPBS+2% HSA and counted.The table below shows the duration of each step in the process and thetotal time elapsed. No further processing was performed on these PBMCsprior to their use in the experiments in this example.

TABLE 24 Elapsed times for steps after blood draw and pooling. Group BGroup C Total Total Time Elapsed Time Elapsed Blood draw &  9:00- 10:00pooling 1^(st) Sepax run 10:08-11:16  2 hr 16 min 10:30-11:45  1 hr 45min 2^(nd) Sepax run 11:21-12:30  3 hr 30 min 11:48-13:04  3 hr 4 minCell counting 12:40-13:00  4 hr 0 min 13:11-13:35  3 hr 35 min Dispenseinto 13:55-14:03  5 hr 3 min 13:37-13:44  3 hr 44 min bags Mediaaddition 14:06-14:20  5 hr 20 min 13:45-13:58  3 hr 58 min Virusaddition 14:55-15:01  6 hr 1 min 14:00-14:30  4 hr 30 min Transduction15:10-19:10 10 hr 10 min 14:30-18:30  8 hr 30 min Transfer & 19:10-19:3610 hr 36 min 18:30-19:04  8 hr 4 min Pellet 1^(st) wash 19:55-20:10 11hr 10 min 19:14-19:30  9 hr 30 min 2^(nd) wash 20:21-20:36 11 hr 36 min19:35-19:51  9 hr 51 min 3^(rd) wash 20:47-21:02 12 hr 2 min 19:56-20:02 9 hr 2 min Cell counting 21:12-22:10 13 hr 10 min 20:03-20:50 10 hr 50min Transportation 21:58-23:10 14 hr 10 min 21:50-21:25 11 hr 25 minDosing mice 23:12-23:30 14 hr 30 min 21:25-21:30 11 hr 30 min

Transduction Efficiency and Cytokine-Independent Survival/ProliferationIn Vitro of PBMCs Transduced by the Methods Above

2.0×10⁶ PBMCs that included T cells and NK cells were seeded induplicate or triplicate into the wells of a 6 well tissue culture platefor each sample. The plates were centrifuged and each sample wasresuspended in 2 ml of Complete OpTmizer™ CTS™ T-Cell Expansion SFM. Nocytokines were added. The plates were incubated in a standard humidifiedtissue culture incubator at 37° C. and 5% CO₂ for 6 days. Half (1 ml) ofthe cell suspension from each well was removed on day 3 and theremaining cells removed on day 6 for to determine cell numbers, percentviability, and percent transduced cells, which was defined as thepercent of FLAG-Tag+ cells by FACS analysis. Total cell counts on day 6were doubled to account for removing half of the cells on day 3.

Proliferation/Survival and Target Killing of Tumors In Vivo by EffectorPBMCs Transduced by the Methods Above

A xenograft model using NSG, or NOD Scid Gamma mice was chosen to probethe ability of human PBMCs transduced with F1-1-27, F1-1-228, F1-1-228U,F1-3-219, and F1-3-219U to survive, proliferate, and kill cognateantigen-expressing tumors in vivo. NSG is a strain of mice that lackmature T cells, NK cells, and B cells and is among the mostimmunodeficient mouse strain described to date. Removal of thesecellular components of the immune system is typically performed toenable human PBMCs to engraft without innate, humoral or adaptive immunereactions from the host. Concentrations of homeostatic cytokinesnormally present only after radiation or lymphodepleting chemotherapy inhumans is achieved due to the absence of the murine extracellular commongamma chain, which enables adoptively transferred human cells to receivesuch cytokines. At the same time, these animals can also be utilized toengraft tumor xenograft targets to examine the efficacy of CARs to killtarget-expressing tumors. While the presence of xenoreactive T cellreceptor antigens in the effector cellular product will eventually giverise to graft versus host disease, these models enable short termevaluation of animal pharmacology and acute tolerability.

Raji cells (ATCC, Manassas, VA) which express endogenous human CD19, andCHO cells (ATCC, Manassas, VA) transfected to stably express human ROR2(CHO-ROR2) were utilized to provide antigen to stimulate the CAReffector cells and to generate uniform target tumors to determine theefficacy of CAR effector cells to kill cognate antigen-expressingtumors. The Raji cells and transgenic CHO variants grew rapidly withdisseminated malignancy after subcutaneous administration into NSG micein combination with Matrigel artificial basement membrane.

Mice were handled in accordance with Institutional Animal Care and UseCommittee approved protocols. Subcutaneous (sc) tumor xenografts wereestablished in the hind flank of female NOD-Prkd^(scid)Il2rg^(tm1)/Begen(B-NSG) mice (Beijing Biocytogen Co. Ltd.). Briefly, cultured Raji cellsand cultured CHO-ROR2 cells were separately washed in DPBS (ThermoFisher), counted, resuspended in cold DPBS and mixed with an appropriatevolume of Matrigel ECM (Corning; final concentration 5 mg/mL) at aconcentration of 0.5×10⁶ cells/200 μl on ice. Animals were prepared forinjection using standard approved anesthesia with hair removal (Nair)prior to injection. 200 μl of either cell suspension in ECM was injectedsc into the rear flanks of 9 or 10 week old mice for Raji and CHO-ROR2cells, respectively.

5 days after tumor inoculation, mice bearing CHO-ROR2 tumors, whichaveraged 77 mm³ in volume, were dosed intravenously (IV) by tail veininjection as follows: NSG mice in Group A received 1×10⁷ PBMCstransduced with F1-1-27 lentiviral particles in 200 μl DPBS (n=4), or200 μl DPBS alone (n=2); NSG mice in Group B received 0.85×10⁷ PBMCstransduced with F1-1-228 lentiviral particles in 200 μl DPBS (n=2),0.85×10⁷ PBMCs transduced with F1-1-228U lentiviral particles in 200 μlDPBS (n=2), or 200 μl DPBS alone (n=2). Similarly, 5 days after tumorinoculation, mice in Group C bearing Raji tumors, which averaged 76 mm³in volume, were dosed intravenously (IV) by tail vein injection with 200μl DPBS alone (n=4) or 1×10⁷ PBMCs transduced with either F1-3-219lentiviral particles in 200 μl DPBS (n=3) or F1-3-219U lentiviralparticles in 200 μl DPBS (n=3). Note that the protocol for theadvantageously fast transduction of resting lymphocytes prior to ex vivoactivation and without ex vivo cell expansion was used to transduce thePBMCs with F1-1-228, F1-1-228U, F1-3-219, and F1-3-219U used for dosingthese mice. The total time elapsed from whole human blood collection toIV dosing of the mice with transduced PBMCs was 14.5 hours for F1-1-228and F1-1-228U, and 11.5 hours for F1-3-219 and F1-3-219U.

Tumors were measured using calipers 2 times a week and tumor volume wascalculated using the following equation: (longest diameter*shortestdiameter²)/2. Approximately 100 μl of blood was collected from eachmouse on days 7, 14, and 21 for analysis by FACS and qPCR. Blood,spleen, and tumor was also collected when the mice were euthanizedconsistent with necropsy guidelines from tumor burden.

Flow Cytometry

For cells harvested from in vitro culture—cells were spun down andresuspended in 0.5 ml FACS Staining Buffer (554656, BD). 2.5 μl human FcBlock (BD, 564220) was added to each sample and incubated at roomtemperature for 10 minutes. Cells were stained with 0.5 μl anti-FLAG TagPE ((anti-DYKDDDDK) 637310, Biolegend) and 0.5 μl Live/Dead FixableGreen Dead cell stain (L34970, Thermo Fisher) for 30 mins on ice. Cellswere washed twice in FACS buffer, fixed in a 1:1 mixture of the FACSbuffer and BD Cytofix (554655, BD), processed with Novocyte (ACEA), andthe resulting data was analyzed with NovoExpress software (ACEA) usinglive gates based on forward and side scatter and the live dead stain.Transduced lymphocytes were measured as FLAG Tag+ cells.

For cells obtained from blood—red blood cells in the freshly collectedblood were lysed using Lysing Buffer (555899, BD) and the remainingcells resuspended in 100 μl of FACS Staining Buffer. 2.5 μl human FcBlock (BD, 564220) was added to each sample and incubated at roomtemperature for 10 minutes. Cells were stained withbiotinylated-cetuximab for 30 mins on ice. Stained cells were washedwith FACS buffer and further stained with 5 μl anti-human CD45-PE-Cy7and 0.5 μl anti-mouse CD45-FITC. 0.4 μl SA-PE was added to samples frommice dosed with cells transduced with F1-1-27, F1-1-228, F1-1-228U, andPBS controls for these groups. 1 μl anti-FLAG Tag PE ((anti-DYKDDDDK)637310, Biolegend) was added to samples from mice dosed with cellstransduced with F1-3-219 and F1-3-219U and PBS controls for this group.Cells were incubated for 30 mins on ice, washed twice in FACS buffer,and resuspended in 100 μl FACS Staining Buffer with 1 μl 7-AAD (420404,Biolegend). Freshly stained samples were processed with Novocyte (ACEA),and the resulting data was analyzed with NovoExpress software (ACEA)using live gates based on forward and side scatter, a live gate based on7-AAD, human CD45+ and examined for the expression of FLAG or eTag.

qPCR

Genomic DNA (gDNA) isolated from blood samples were evaluated for thepresence of transduced lymphocytes by bioanalytical qPCR. Genomic DNAwas isolated from 50 μl blood samples using the QIAamp DNA Blood Minikit (Qiagen 51106) and the DNA was further cleaned using the QIAamp DNAMicro Kit (56304). A TaqMan assay (Thermo Fisher) was performed on theisolated genomic DNA using a primer and probe set specific for the 5′LTR of lentivirus to detect transduced cells.

Results

Lentiviral particles pseudotyped with VSV-G and encoding a CAR and alymphoproliferative element were used in this experiment. Lentiviralparticles F1-1-228 and F1-1-228U encoded a MRB-CAR to ROR2 andlentiviral particles F1-2-219 and F1-3-219U encoded a CAR to CD19.Lentiviral particles F1-1-228U and F1-3-219U also displayedUCHT1scFvFc-GPI on their surface. PBMCs were isolated from human bloodby density gradient centrifugation with Ficoll-Paque™. The fresh PBMCswere transduced with the lentiviral particles in standard blood bagswithout prior activation of the cells ex vivo. After a 4 hourtransduction, the cells were washed and used in the experiments below. Askilled artisan will understand that the entire process from bloodcollection to washed cells could be performed in a closed system. As acontrol, PBMCs were activated overnight, transduced with lentiviralparticles encoding a CAR without a lymphoproliferative element orUCHT1scFvFc-GPI, and expanded ex vivo for 15 days (F1-1-27).

Transduced PBMCs were cultured in vitro in the absence of cytokines.FIG. 37 shows transduction efficiencies at Day 6. UCHT1scFvFc-GPIincreased the transduction efficiency of both F1-1-228 and F1-3-219.Resting PBMCs, when exposed for 4 hours to F1-1-228U or F1-3-219U hadtransduction efficiencies of 34% and 73%, respectively. FIG. 38 showsthe total number of viable cells in these cultures between days 3 and 6.Resting PBMCs transduced with either F1-1-228U or F1-3-219U survived andeven proliferated between day 3 and day 6 in the absence of exogenouscytokines while PBMCs transduced with either F1-1-228 or F1-3-219 didnot. These results demonstrate that retroviral particles displaying anactivation element and encoding a lymphoproliferative element cantransduce resting PBMCs in 4 hours and that these transduced PMBCs canproliferate and survive in vitro when cultured for 6 days in the absenceof exogenous cytokines.

Immunodeficient mice bearing ROR2 or CD19 tumors were dosedintravenously with 1×10⁷ PBMCs that express a CAR to ROR2 or CD19,respectively. The ability of the transduced PBMCs to survive andproliferate in vivo was examined over time. FIGS. 39A-C show thelentiviral copies per μg of genomic DNA by qPCR as a readout fortransduced cells. FIG. 39A shows that lentiviral copies per μg ofgenomic DNA for F1-1-27, which does not encode for a lymphoproliferativeelement, decreased from an average of 884 on day 7 to below the lowerlimit of quantitation (LLOQ) by day 21. FIG. 39B shows that thelentiviral copies for F1-1-228U was below the LLOQ on days 7 and 14, butincreased above the LLOQ by day 21 and increased to an average of 1,939on day 25. FIG. 39C shows that the lentiviral copies for F1-3-219Uincreased from below the LLOQ on day 7 to 20,430 on day 14 when the micewere euthanized. Lentiviral copies in the control samples, F1-1-228 andF1-3-219, remained below the LLOQ. FIG. 40 shows the number oftransduced cells per 200 ul of blood at the time the mice wereeuthanized (the last time points are shown in FIG. 39 ) as determined byFACS analysis for the eTag or FLAG-Tag of the CAR construct. Significantnumbers of CAR+ cells were detected per 200 ul of blood from mice dosedwith cells transduced with F1-1-228U (5,857) and F1-3-219U (121,324).Lymphocytes transduced with F1-1-228 or F1-3-219U killed tumorsexpressing their target antigen in vivo (FIGS. 41A and B). In both casesthe lymphocytes reduced the tumor volume in a delayed manner. Not to belimited by theory, but this delay is consistent with a need for theinjected cells to expand, which took time as seen by qPCR. Later timepoints could not be studied in this experiment because the miceharboring ROR2 tumors reach euthanasia guidelines for tumor burden andthe mice harboring Raji tumors developed graft-versus-host diseasecaused by the significant number of transduced and expanded lymphocytesin the mice. Together these data show that retroviral particlesdisplaying an activation element and encoding a lymphoproliferativeelement can transduce resting PBMCs in 4 hours and that these transducedPMBCs can proliferate and survive in vivo. Both lymphoproliferativeelements tested in this experiment, MycTag 2A Jun-IL13Ra-MPL-CD40 andIL-7-IL-7Rα-IL-2Rβ, displayed the ability to promote survival andproliferation in vivo. Furthermore, these lymphocytes transduced in thismanner expressing a MRB-CAR (F1-1-228U) or a traditional CAR (F1-3-219U)were able to recognize and kill tumor cells in vivo.

Example 23. Exemplary RIR Retroviral Particles Transduce Primary T CellsFollowing Contacting Times as Brief as Less than 1 Minute

In this example, exemplary RIR Retroviral Particles and controlretroviral particles placed in contact with resting primary human Tcells that were PBMCs, which had not been preactivated, for timesbetween 4 hours and less than 1 minute, were assessed for their abilityto transduce the cells and survive and proliferate for up to 6 days inculture in the absence of exogenous cytokines. Four unique replicationincompetent recombinant retroviral particles (“RIR retroviral particles)were examined. Each of the RIR retroviral particles comprised anenvelope polypeptide (e.g., a pseudotyping element) and a polynucleotideencoding a CAR. As shown diagrammatically in the RIR retroviral particleof FIG. 42A, certain retroviral particles further comprised anactivation element on the surface of the retroviral particle and/or asshown in FIG. 42B, the polynucleotide also encoded a lymphoproliferativeelement. As shown in FIG. 43 , RIR retroviral particles “A” did notcomprise either an activation element on the surface of the retroviralparticle or a polynucleotide encoding a lymphoproliferative element. RIRretroviral particles “B” comprised an activation element on the surfacebut not a lymphoproliferative element. RIR retroviral particles “C” didnot comprise an activation element on the surface but did comprise alymphoproliferative element. RIR retroviral particles “D” were RIRretroviral particles that comprised both an activation element on thesurface of the retroviral particle and a polynucleotide encoding alymphoproliferative element. Following the contacting between the PBMCsand the RIR retroviral particles, the cells were washed and placed inculture. No exogenous cytokines were added to the samples at any pointin this example. Transduction efficiency was examined on day 6.

Methods

The RIR retroviral particles made and used in this experiment, werereplication incompetent recombinant lentiviral particles that wereproduced in 293T cells (Lenti-X™ 293T, Clontech) that were adapted tosuspension culture in Freestyle™ 293 Expression Medium (Thermo FisherScientific), which is serum-free chemically defined medium. The cellswere transiently transfected using PEI with a 3^(rd) generationlentiviral genomic plasmid and separate packaging plasmids encodinggag/pol, rev, and a pseudotyping plasmid encoding VSV-G. For RIRretroviral particles B and D, a plasmid encoding the activation element,anti-CD3(UCHT1)scFvFc-GPI, was also included in the transfection.

The genomic plasmids for retroviral particles A and B included an EF-1apromoter, operatively linked to a polynucleotide encoding one engineeredsignaling polypeptide: a first signaling polypeptide comprising a CARcontaining a FLAG tag, an anti-CD19 ASTR, a CD8 transmembrane domain,and a CD3z intracellular signaling domain. The genomic plasmids for RIRretroviral particles C and D also included the EF-1a promoter, but itwas operatively linked to a polynucleotide encoding two engineeredsignaling polypeptides: a first signaling polypeptide comprising thesame CAR as that encoded in RIR retroviral particles A and B, followedby a T2A ribosomal skip sequence; and a second engineered signalingpolypeptide that included a constitutively active lymphoproliferativeelement comprising a cytokine receptor polypeptide that is known toactivate JAK pathways. A diagram that shows the transcriptional unitencoding the first and second engineered signaling polypeptides of RIRretroviral particles C and D is shown in FIG. 42B.

On Day 0, PBMCs were enriched from buffy coats (San Diego Blood Bank) bydensity gradient centrifugation with Ficoll-Paque PREMIUM® (GEHealthcare Life Sciences) and SepMate™-50 (Stemcell™ Technologies)according to the manufacturer's instructions. No additional steps weretaken to remove monocytes. After isolation, the PBMCs were diluted to1×10⁶ PBMCs per 1 ml of X-Vivo15 (LONZA) and 1 ml was seeded into eachwell of 96 deep-well plates. No anti-CD3, anti-CD28, IL-2, IL-7, orother exogenous cytokine was added to activate or otherwise stimulatethe lymphocytes prior to transduction. Lentiviral particles were addeddirectly to the non-stimulated PBMCs at an MOI of 1. The transductionswere incubated at 37° C. and 5% CO₂ for either 4 hours, 2 hours, 30minutes, 15 minutes, 7.5 minutes, 5 minutes, 2.5 minutes or notincubated at all before the cells were washed out of the transductionmix 3 times in DPBS+2% HSA. The cells in each well were then resuspendedin 1 ml X-Vivo15 and incubated at 37° C. and 5% CO₂. For samples treatedwith antiviral drugs, dapivirine or dolutegravir was added to a finalconcentration of 10 μM during the transduction and the transductionreaction was incubated at 37° C. and 5% CO₂ for 4 hours. The drugs werereplenished at the same concentrations in the recovery medium after thethree washes. No exogenous cytokines were added to the samples at anytime. Samples were collected at Day 6 and transduction efficienciesbased on FLAG expression were determined by FACS analysis using alymphocyte gate based on forward and side scatter.

Results

FIG. 43 shows the number of transduced cells (CD3+FLAG+) per μl at Day 6after transduction of unstimulated PBMCs by the different recombinantRIR retroviral particles for the indicated contacting time. At all timepoints, the transduction efficiency of RIR retroviral particles B and D,which comprised an activation element on the surface of the retroviralparticle, was greater than the transduction efficiency of theircounterpart RIR retroviral particles A and C, respectively, that lackedan activation element. Furthermore, at all time points, the number of Tcells transduced with RIR retroviral particles D, which comprised bothan activation element on the surface of the retroviral particle and apolynucleotide encoding a lymphoproliferative element, was greater thanthe number of PBMCs transduced with RIR retroviral particles B whichwere identical except that they lacked a polynucleotide encoding alymphoproliferative element. It was unexpected and is particularlynoteworthy that RIR retroviral particles D were efficient in transducingPBMCs following a short 4 hour or less contacting time down tocontacting times of less than 1 minute, and promoting survival andproliferation of transduced cells in the absence of exogenous growthfactors for at least 6 days. FIG. 43 also shows that the inhibition oftransduction by dapivirine, a reverse transcriptase inhibitor, anddolutegravir, an integrase inhibitor, demonstrate that geneticmodification and transgene expression by these PBMCs is notpseudotransduction, but rather is the result of transduction in whichthe viral transgene RNA is reverse transcribed, integrated into thegenomes of PBMCs, and expressed. Thus, exemplary RIR retroviralparticles comprising an envelope polypeptide (e.g., pseudotypingelement), a cytokine receptor polypeptide lymphoproliferative element, aCAR, and an activation element, have unique properties that endow themwith the ability to transduce primary T cells following very briefcontacting times and without preactivation, and promote survival andproliferation of those cells in the absence of exogenous growth factorsfor at least 6 days.

Example 24. Exemplary RIR Retroviral Particles Transduce Primary T CellsFollowing a 4-Hour Contacting Time without Preactivation and Bestow Uponthese Modified Cells, the Ability to Proliferate and Survive In Vitro inthe Absence of Exogenous Cytokines for 21 Days

In this example, exemplary RIR retroviral particles and control RIRretroviral particles placed in contact with resting primary human Tcells that were PBMCs, which had not been preactivated, for 4 hours,were assessed for their ability to transduce the cells and promote thesurvival and/or proliferation of the PBMCs when they were cultured invitro in the absence of exogenous added cytokines for 21 days. Therecombinant retroviral particles (“RIR retroviral particles”) used inthis example were the same recombinant lentiviral particles B, C, and Dused in Example 23 (See FIG. 44 ). Donor matched PBMCs were added to thecultures to provide CD19+ B-cell activation of the CAR, which wasdirected to CD19. No exogenous cytokines were added to the samples atany point in this example.

Methods

On Day 0, PBMCs were enriched from Leukocyte Reduction System WhiteBlood Cells (LRS-WBCs) (San Diego Blood Bank) by density gradientcentrifugation with Ficoll-Paque PREMIUM® (GE Healthcare Life Sciences)according to the manufacturer's instructions followed by lysis of redblood cells. No additional steps were taken to remove monocytes. Aportion of the PBMCs were frozen at 2×10⁷ PBMCs per vial for later usein feeding the CD19-CAR expressing cells. The remaining PBMCs werediluted to 1.0×10⁶ viable cells/ml in Complete OpTmizer™ CTS™ T-CellExpansion SFM and 5 ml were added to a 50 ml Conical tube for eachsample. No anti-CD3, anti-CD28, IL-2, IL-7, or other exogenous cytokinewas added to activate or otherwise stimulate the lymphocytes prior totransduction. On-test RIR retroviral particles were added directly tothe non-stimulated PBMCs in conical tubes at an MOI of 1 and incubatedfor 4 hours at 37° C. and 5% CO₂. The cells were then washed 3 times inDPBS+2% HSA before being resuspended in 30 ml Complete OpTmizer™ CTS™T-Cell Expansion SFM and transferred to wells of G-Rex® 6-well gaspermeable cell culture devices (Wilson Wolf). The cells from each wellwere collected on Day 7 to determine cell numbers, percent viability,and percent transduced cells, which was defined as the percent ofFLAG-Tag+ cells by FACS analysis using a lymphocyte gate. Half of thecells from each transduction were reseeded into the wells of a G-Rex®6-well plate. Previously cryopreserved Day 0 donor matched PBMCs wereadded to each sample at a ratio of 1 transduced cell to 1 Day 0 donormatched CD19+ B cell (the percentage of CD19+ B cells in the PBMCs wasdetermined by FACs on Day 0). Complete OpTmizer™ CTS™ T-Cell ExpansionSFM was then added to each well to bring the volume to 30 ml and thetransduced cells were incubated at 37° C. and 5% CO₂. On Day 14, 15 mlof spent media was replaced with fresh media and additional Day 0 PBMCswere added at the same 1:1 ratio. The cells were assayed again on days14 and 21 to determine cell numbers, percent viability, and theexpression of surface markers by FACS.

Results

FIG. 44 shows the number of transduced PBMCs as measured by FACS for CARexpression (by FLAG tag) after 7, 14, and 21 days of culture in vitro inthe absence of exogenous cytokines. PBMCs that were contacted for 4hours with RIR retroviral particles D were efficiently transduced andexpanded in the absence of cytokines. Fewer CAR+ cells were observed incultures transduced with either RIR retroviral particles B or C at day 7and the cell numbers declined over time in both cases. Thus, RIRretroviral particles D comprising an envelope polypeptide (e.g., apseudotyping element), a cytokine receptor polypeptidelymphoproliferative element, a CAR, and an activation element (FIG. 42A)have unique properties that endow them with the ability to transduceprimary T cells following a 4-hour contacting time withoutpreactivation, and bestow upon these transduced cells, the ability toproliferate and/or survive in the absence of exogenous cytokines.

Example 25. Exemplary RIR Retroviral Particles Transduce Primary T CellsRapidly without Preactivation, and Bestow Upon these Modified Cells, InVivo Proliferation and Survival Properties, Thereby Exemplifying a RapidPoint-of-Care (rPOC) Cell Manufacturing Process

In this example, replication incompetent recombinant retroviralparticles (“RIR retroviral particles) placed in contact with restingprimary human T cells that were PBMCs, which had not been preactivated,for 4 hours were assessed for their ability to transduce the cells andpromote expansion, engraftment, and/or persistence of the lymphocytes invivo in a murine model without an ex vivo manufacturing step to expandthe transduced PBMCs. Three unique RIR retroviral particles wereexamined. Each of the RIR retroviral particles were lentivirus particlesthat comprised a pseudotyping element and a polynucleotide encoding achimeric antigen receptor (“CAR”). RIR retroviral particles “E” alsocomprised both an activation element on the surface of the retroviralparticle and a polynucleotide encoding a lymphoproliferative element.RIR retroviral particles “F” did not comprise an activation element onthe surface, but did comprise a polynucleotide encoding alymphoproliferative element. RIR retroviral particles “G” did notcomprise either an activation element on the surface of the retroviralparticle or a polynucleotide encoding a lymphoproliferative element.Unlike PBMCs transduced with RIR retroviral particles E and F thatunderwent rapid cell processing, PBMCs transduced with RIR retroviralparticles G underwent a traditional activation, transduction, and exvivo cell manufacturing process that took 15 days. PBMCs modified witheach RIR retroviral particle were administered intravenously to mice,and the ability of the transduced cells to proliferate and survive wasexamined in the blood after 7, 14, and 21 days.

Methods

The RIR retroviral particles made and used in this experiment, wererecombinant lentiviral particles that were produced in 293T cells(Lenti-X™ 293T, Clontech) that were adapted to suspension culture inFreestyle™ 293 Expression Medium (Thermo Fisher Scientific), which isserum-free chemically defined medium. The cells were transientlytransfected using PEI with a 3^(rd) generation lentiviral genomicplasmid and separate packaging plasmids encoding gag/pol, rev, and apseudotyping plasmid encoding VSV-G. For retroviral particles E, aplasmid encoding the activation element anti-CD3(UCHT1)scFvFc-GPI wasalso included in the transfection. The genomic plasmids for RIRretroviral particles E and F were identical and included an EF-1apromoter, operatively linked to a polynucleotide encoding two engineeredsignaling polypeptides: a first signaling polypeptide that was a CARcontaining a FLAG tag, an anti-ROR2 MRB ASTR, a CD8a stalk andtransmembrane domain, a CD137 intracellular domain, and a CD3zintracellular signaling domain, followed by a T2A ribosomal skipsequence; and a second engineered signaling polypeptide that included aconstitutively active lymphoproliferative element comprising a cytokinereceptor polypeptide that is known to activate JAKs. The genomic plasmidfor RIR retroviral particle G was identical to E and F except that G didnot encode the T2A sequence or the lymphoproliferative element.

Traditional Activation, Transduction, and Ex Vivo Cell ManufacturingProcess (Processing Time—15 Days)

On Day 0, PBMCs were isolated from anticoagulant-citrate-dextrose (ACD)peripheral blood from a healthy volunteer with informed consent bydensity gradient centrifugation with Ficoll-Pacque™ (General Electric)using a CS-900.2 kit (BioSafe; 1008) on a Sepax 2 S-100 device (Biosafe;14000) according to the manufacturer's instructions. 3.0×10⁷ viablePBMCs were seeded in a 1 L G-Rex (Wilson-Wolf) and the volume wasbrought to 60 ml with Complete OpTmizer™ CTS™ T-Cell Expansion SFMsupplemented with 100 IU/ml IL-2 (Novoprotein, GMP-CD66), 10 ng/ml IL-7(Novoprotein, GMP-CD47), and 50 ng/ml anti-CD3 antibody (OKT3,Novoprotein) to activate the PBMCs, which included T cells and NK cells,for viral transduction. After incubation overnight at 37° C. and 5% CO₂,RIR retroviral particles G were added directly to the activated PBMCs atan MOI of 5 (440 l) and incubated overnight at 37° C. and 5% CO₂.Following the overnight incubation, the total volume of media in theG-Rex was increased to 100 ml with Complete OpTmizer™ CTS™ T-CellExpansion SFM supplemented with NAC (Sigma) to increase the finalconcentration by 10 mM. rhIL-2 and rhIL-7 were also added to bring thefinal concentrations to 100 IU/ml and 10 ng/ml, respectively. The G-Rexdevice was incubated in a standard humidified tissue culture incubatorat 37° C. and 5% CO₂ with additions of 100 IU/ml recombinant human IL-2and 10 ng/ml recombinant human IL-7 solution every 48 hours. The cellswere expanded through day 15 before being harvested. These transducedcells were washed in freezing media (70% RPMI 1640, 20% heat-inactivatedFBS, 10% DMSO) and cryopreserved in 1 ml aliquots at 5.0×10⁷ cells/mlfor later use. Two days prior to use in the experiments in this examplebelow, the cells were thawed and rested for 2 days in a G-Rex100Mcontaining 377 ml Complete OpTmizer™ CTS™ T-Cell Expansion SFM(OpTmizer™ CTS™ T-Cell Expansion Basal Medium 1 L (Thermo Fisher,A10221) supplemented with 26 ml OpTmizer™ CTS™ T-Cell ExpansionSupplement (Thermo Fisher, A10484-02), 25 ml CTS™ Immune Cell SR (ThermoFisher, A2596101), and 10 ml CTS™ GlutaMAX™-I Supplement (Thermo Fisher,A1286001) according to manufacturer's instructions) supplemented with100 IU/ml of IL-2 (Novoprotein), 10 ng/ml IL-7 (Novoprotein), andsufficient NAC to increase the final concentration by 10 mM.

Rapid Point-of-Care (rPOC) Cell Manufacturing Process without Ex VivoCell Expansion (Processing Time—Less than 15 Hours)

PBMCs were isolated from ACD peripheral blood from a healthy volunteerwith informed consent by density gradient centrifugation withFicoll-Pacque™ (General Electric) using a CS-900.2 kit (BioSafe; 1008)on a Sepax 2 S-100 device (Biosafe; 14000) according to themanufacturer's instructions. 5.5×10⁷ freshly enriched, viable PBMCs wereseeded into each of 2 standard blood collection bags and the volume wasbrought to 55 ml with Complete OpTmizer™ CTS™ T-Cell Expansion SFM suchthat the cells were at a density of 1.0×10⁶/ml. No anti-CD3, anti-CD28,IL-2, IL-7, or other exogenous cytokine was added to activate orotherwise stimulate the lymphocytes ex vivo prior to transduction.On-test RIR retroviral particles were added directly to thenon-stimulated PBMCs in the blood collection bags at an MOI of 1 withRIR retroviral particles E going into one bag and RIR retroviralparticles F going into the other bag. The transduction reaction mixtureswere gently massaged to mix the contents then incubated for 4 hours inthe blood collection bags in a standard humidified tissue cultureincubator at 37° C. and 5% CO₂. The PBMCs from each bag were thentransferred to a 50 ml Conical tube and washed 3 times in DPBS+2% HSAbefore being resuspended in 5 mils DPBS+2% HSA and counted. No furtherprocessing was performed on these PBMCs prior to their use in theexperiments in this example.

Dosing, In Vivo Proliferation, and Survival of PBMCs Transduced by theMethods Above

Mice were handled in accordance with Institutional Animal Care and UseCommittee approved protocols. The ability of PBMCs transduced with RIRretroviral particles E, F, and G to proliferate and survive in vivo wasassessed in female NOD Scid Gamma mice (B-NSG, Beijing Biocytogen Co.Ltd.) with established CHO cell tumors stably transfected to expresshuman ROR2 and suspended in Matrigel ECM, in their right hind flank. 5days after tumor inoculation, mice bearing CHO-ROR2 tumors, whichaveraged 77 mm³ in volume, were dosed intravenously (IV) by tail veininjection as follows: 2 mice received 0.85×10⁷ PBMCs transduced withretroviral particles “E” in 200 μl DPBS, 2 mice received 0.85×10⁷ PBMCstransduced with retroviral particles “F” in 200 μl DPBS, and 4 micereceived 1.0×10⁷ PBMCs transduced with retroviral particles “G” in 200μl DPBS. Note that the rPOC protocol for the rapid transduction oflymphocytes without prior activation and without ex vivo cell expansion,was used to transduce the PBMCs with retroviral particles E and F usedfor dosing these mice. The total time elapsed from whole human bloodcollection to IV dosing of the mice with modified PBMCs was 14.5 hoursfor retroviral particles E and F. Approximately 100 μl of blood wascollected from each mouse on days 7, 14, 21, and 25 for analysis byqPCR.

qPCR

Genomic DNA (gDNA) isolated from blood samples was evaluated for thepresence of transduced lymphocytes by bioanalytical qPCR. Genomic DNAwas isolated from 50 μl blood samples using the QIAamp DNA Blood Minikit (Qiagen 51106) and the DNA was further cleaned using the QIAamp DNAMicro Kit (56304). A TaqMan assay (Thermo Fisher) was performed on theisolated genomic DNA using a primer and probe set specific for the 5′LTR of lentivirus to detect transduced cells.

Results

The ability of the transduced PBMCs to proliferate and survive in vivoin mice was examined in the blood after 7, 14, 21, and 25 days. Thelegend for FIGS. 45A and 45B indicates the different components thatwere included in RIR retroviral particles E, F and G, as well as themanufacturing process (rapid point of care (rPOC) or 15-day traditional(T)) used to process cells for each of the samples. FIGS. 45A and 45Bshows the lentiviral copies per μg of genomic DNA isolated from blood ofthe MRB-ROR2 CAR-expressing PBMC dosed mice, by qPCR as a readout forthe number of transduced cells. FIG. 45A shows the persistence oftransduced PBMCs manufactured using the rapid point-of-care (rPOC)process with RIR retroviral particles E and F, as indicated, without exvivo cell expansion. The lentiviral copies in mice dosed with PBMCstransduced with RIR retroviral particles E, which comprised apseudotyping element, a cytokine receptor polypeptidelymphoproliferative element, a CAR, and an activation element, was belowthe lower limit of quantitation (LLOQ) on days 7 and 14, but increasedabove the LLOQ by day 21 and increased to an average of 1,939 on day 25.The lentiviral copies in mice dosed with PBMCs transduced with RIRretroviral particles F, which were identical to retroviral particles Ebut for the lack of an activation element on the surface of retroviralparticles E, remained below the LLOQ.

FIG. 45B analyzes the persistence of transduced PBMCs manufactured usinga traditional protocol of activation, transduction, and ex vivo cellmanufacturing process with retroviral particles G. These RIR retroviralparticles did not have an activation element on their surface, and didnot have a polynucleotide encoding a lymphoproliferative element. Thelentiviral copies in mice dosed with PBMCs transduced with retroviralparticles G decreased from an average of 884 on day 7 to below the lowerlimit of quantitation (LLOQ) by day 21. Overall this experimentdemonstrates that RIR retroviral particles E comprising an envelopepolypeptide (e.g., a pseudotyping element), a cytokine receptorpolypeptide lymphoproliferative element, a CAR, and an activationelement have unique properties that endow them with the ability totransduce PBMCs following a 4-hour contacting time withoutpreactivation, and bestow upon these transduced cells, the ability toproliferate and survive in an in vivo mouse model. Furthermore, such invivo persistence was a substantial improvement to that seen with PBMCstransduced with retroviral particles encoding an identical CAR producedusing a traditional activation, transduction, and ex vivo cellmanufacturing process.

The disclosed embodiments, examples and experiments are not intended tolimit the scope of the disclosure or to represent that the experimentsbelow are all or the only experiments performed. Efforts have been madeto ensure accuracy with respect to numbers used (e.g., amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. It should be understood that variations in the methods asdescribed may be made without changing the fundamental aspects that theexperiments are meant to illustrate.

Those skilled in the art can devise many modifications and otherembodiments within the scope and spirit of the present disclosure.Indeed, variations in the materials, methods, drawings, experiments,examples, and embodiments described may be made by skilled artisanswithout changing the fundamental aspects of the present disclosure. Anyof the disclosed embodiments can be used in combination with any otherdisclosed embodiment.

In some instances, some concepts have been described with reference tospecific embodiments. However, one of ordinary skill in the artappreciates that various modifications and changes can be made withoutdeparting from the scope of the invention as set forth in the claimsbelow. Accordingly, the specification and figures are to be regarded inan illustrative rather than a restrictive sense, and all suchmodifications are intended to be included within the scope of invention.

TABLE 6 Codes, names, cytokine receptors, ITAM motif, andamino acid sequences for parts P1-P2, P1, P2, P3, and P4. Code NameAmino Acid Sequence M001 eTAG IL7RA InsMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPPPCL (interleukin 7QELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNreceptor)KNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPEINNSSGEMDPILLPPCLTISILSFFSVALLVILACVL (SEQID NO: 292) M002 eTAG IL7RA InsMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPPPCL (interleukin 7QELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNreceptor)KNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQPEINNSSGEMDPILLPPCLTISILSFFSVALLVILACVL(SEQ ID NO: 293) M007 Myc Tag LMP1MEQKLISEEDLEHDLERGPPGPRRPPRGPPLSSSLGLALLLLLLALLFWLYIVMSDWTGGALLVLYSFALMLIIIILNC_007605_1IIFIFRRDLLCPLGALCILLLMITLLLIALWNLHGQALFLGIVLFIFGCLLVLGIWIYLLEMLWRLGATIWQLLAFFLAFFLDLILLIIALYLQQNWWTLLVDLLWLLLFLAILIWM (SEQ ID NO: 294) M008 Myc LMP1MEQKLISEEDLSSSLGLALLLLLLALLFWLYIVMSDWTGGALLVLYSFALMLIIIILIIFIFRRDLLCPLGALCILLLNC_007605_1MITLLLIALWNLHGQALFLGIVLFIFGCLLVLGIWIYLLEMLWRLGATIWQLLAFFLAFFLDLILLIIALYLQQNWWTLLVDLLWLLLFLAILIWM (SEQ ID NO: 295) M009 LMP1MEHDLERGPPGPRRPPRGPPLSSSLGLALLLLLLALLFWLYIVMSDWTGGALLVLYSFALMLIIIILIIFIFRRDLLCNC_007605_1PLGALCILLLMITLLLIALWNLHGQALFLGIVLFIFGCLLVLGIWIYLLEMLWRLGATIWQLLAFFLAFFLDLILLIIALYLQQNWWTLLVDLLWLLLFLAILIWM (SEQ ID NO: 296) M010 LMP1MSLGLALLLLLLALLFWLYIVMSDWTGGALLVLYSFALMLIIIILIIFIFRRDLLCPLGALCILLLMITLLLIALWNLNC_007605_1HGQALFLGIVLFIFGCLLVLGIWIYLLEMLWRLGATIWQLLAFFLAFFLDLILLIIALYLQQNWWTLLVDLLWLLLFLAILIWM (SEQ ID NO: 297) M012 eTAG CRLF2MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPtranscript variantQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGN1 NM_022148_3KNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGAETPTPPKPKLSKCILISSLAILLMVSLLLLSLW (SEQ IDNO: 298) M013 eTAG CRLF2MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPtranscript variantQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGN1 NM_022148_3KNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQAETPTPPKPKLSKCILISSLAILLMVSLLLLSLW (SEQID NO: 299) M018 eTAG CSF2RBMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPNM_000395_2QELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGTESVLPMWVLALIEIFLTIAVLLAL (SEQ ID NO: 300)M019 eTAG CSF2RBMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPNM_000395_2QELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQTESVLPMWVLALIEIFLTIAVLLAL (SEQ IDNO: 301) M024 eTAG CSF3RMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPtranscript variantQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGN1 NM_000760_3KNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGTPEGSELHIILGLFGLLLLLNCLCGTAWLCC (SEQ IDNO: 302) M025 eTAG CSF3RMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPtranscript variantQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGN1 NM_000760_3KNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQTPEGSELHIILGLFGLLLLLNCLCGTAWLCC (SEQ IDNO: 303) M030 eTAG EPORMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPtranscript variantQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGN1 NM_000121_3KNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGTPSDLDPCCLTLSLILVVILVLLTVLALLS (SEQ ID NO: 304)M031 eTAG EPORMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPtranscript variantQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGN1 NM_000121_3KNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQTPSDLDPCCLTLSLILVVILVLLTVLALLS (SEQ IDNO: 305) M036 eTAG GHRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPtranscript variantQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGN1 NM_000163_4KNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGTLPQMSQFTCCEDFYFPWLLCIIFGIFGLTVMLFVFLFS(SEQ ID NO: 306) M037 eTAG GHRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPtranscript variantQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGN1 NM_000163_4KNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQTLPQMSQFTCCEDFYFPWLLCIIFGIFGLTVMLFVFLFS (SEQ ID NO: 307) M042 eTAG truncatedMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPafter Fn F523CQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNIL27RAKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKNM_004843_3CNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGHLPDNTLRWKVLPGILCLWGLFLLGCGLSLA (SEQ IDNO: 308) M043 eTAG truncatedMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPafter Fn F523CQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNIL27RAKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQHLPDNTLRWKVLPGILCLWGLFLLGCGLSLA (SEQNM_004843_3 ID NO: 309) M048 eTAG truncatedMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPafter Fn S505NQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNMPLKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKNM_005373_2CNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGETATETAWISLVTALHLVLGLNAVLGLLLL (SEQ IDNO: 310) M049 eTAG truncatedMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPafter Fn S505NQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNMPLKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQETATETAWISLVTALHLVLGLNAVLGLLLL (SEQ IDNM_005373_2 NO: 311) E006 eTag 0A JUNMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPNM_002228_3QELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKV(SEQ ID NO: 312) E007 eTag 1A JUNMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPNM_002228_3QELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVA(SEQ ID NO: 313) E008 eTag 2A JUNMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPNM_002228_3QELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVAA(SEQ ID NO: 314) E009 eTag 3A JUNMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPNM_002228_3QELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVAAA (SEQ ID NO: 315) E010 eTag 4A JUNMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPNM_002228_3QELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVAAAA (SEQ ID NO: 316) E011 Myc Tag 0A JUNMTILGTTFGMVFSLLQVVSGEQKLISEEDLLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKV (SEQNM_002228_3 ID NO: 317) E012 Myc Tag 1A JUNMTILGTTFGMVFSLLQVVSGEQKLISEEDLLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVA (SEQNM_002228_3 ID NO: 318) E013 Myc Tag 2A JUNMTILGTTFGMVFSLLQVVSGEQKLISEEDLLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVAANM_002228_3 (SEQ ID NO: 319) E014 Myc Tag 3A JUNMTILGTTFGMVFSLLQVVSGEQKLISEEDLLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVAAANM_002228_3 (SEQ ID NO: 320) E015 Myc Tag 4A JUNMTILGTTFGMVFSLLQVVSGEQKLISEEDLLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVAAAANM_002228_3 (SEQ ID NO: 321) T001 CD2 transcriptLIIGICGGGSLLMVFVALLVFYI (SEQ ID NO: 322) variant 1 NM_001328609_1 T002CD3D transcript GIIVTDVIATLLLALGVFCFA (SEQ ID NO: 323) variant 1NM_000732_4 T003 CD3E VMSVATIVIVDICITGGLLLLVYYWS (SEQ ID NO: 324)NM_000733_3 T004 CD3G GFLFAEIVSIFVLAVGVYFIA (SEQ ID NO: 325) NM_000073_2T005 CD3Z CD247 LCYLLDGILFIYGVILTALFL (SEQ ID NO: 326)transcript variant 1 NM_198053_2 T006 CD4 transcriptMALIVLGGVAGLLLFIGLGIFF (SEQ ID NO: 327) variant 1 and 2 NM_000616_4 T007CD8A transcript IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 328) variant 1NM_001768_6 T008 CD8B transcriptLGLLVAGVLVLLVSLGVAIHLCC (SEQ ID NO: 329) variant 2 NM_172213_3 T009 CD27ILVIFSGMFLVFTLAGALFLH (SEQ ID NO: 330) NM_001242_4 T010 CD28 transcriptFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 331) variant 1 NM_006139_3 T011CD40 transcript ALVVIPIIFGILFAILLVLVFI (SEQ ID NO: 332) variant 1 and 6NM_001250_5 T012 CD79A transcriptIITAEGIILLFCAVVPGTLLLF (SEQ ID NO: 333) variant 1 NM_001783_3 T013CD79B transcript GIIMIQTLLIILFIIVPIFLLL (SEQ ID NO: 334) variant 3NM_001039933_2 T014 CRLF2 transcriptFILISSLAILLMVSLLLLSLW (SEQ ID NO: 335) variant 1 NM_022148_3 T015CRLF2 transcript CILISSLAILLMVSLLLLSLW (SEQ ID NO: 336) variant 1NM_022148_3 T016 CSF2RA transcriptNLGSVYIYVLLIVGTLVCGIVLGFLF (SEQ ID NO: 337) variant 7 and 8NM_001161529_1 T017 CFS2RB MWVLALIVIFLTIAVLLAL (SEQ ID NO: 338)NM_000395_2 T018 CFS2RB MWVLALIEIFLTIAVLLAL (SEQ ID NO: 339) NM_000395_2T019 CSF3R transcript IILGLFGLLLLLTCLCGTAWLCC (SEQ ID NO: 340) variant 1NM_000760_3 T020 CSF3R transcriptIILGLFGLLLLLNCLCGTAWLCC (SEQ ID NO: 341) variant 1 NM_000760_3 T021EPOR transcript LILTLSLILVVILVLLTVLALLS (SEQ ID NO: 342) variant 1NM_000121_3 T022 EPOR transcriptCCLTLSLILVVILVLLTVLALLS (SEQ ID NO: 343) variant 1 NM_000121_3 T023FCER1G LCYILDAILFLYGIVLTLLYC (SEQ ID NO: 344) NM_004106_1 T024 FCGR2CIIVAVVTGIAVAAIVAAVVALIY (SEQ ID NO: 345) NM_201563_5 T025FCGRA2 transcript IIVAVVIATAVAAIVAAVVALIY (SEQ ID NO: 346) variant 1NM_001136219_1 T026 GHR transcriptFPWLLIIIFGIFGLTVMLFVFLFS (SEQ ID NO: 347) variant 1 NM_000163_4 T027GHR transcript FPWLLCIIFGIFGLTVMLFVFLFS (SEQ ID NO: 348) variant 1NM_000163_4 T028 ICOS FWLPIGCAAFVVVCILGCILI (SEQ ID NO: 349) NM_012092.3T029 IFNAR1 IWLIVGICIALFALPFVIYAA (SEQ ID NO: 350) NM_000629_2 T030IFNAR2 transcript IGGIITVFLIALVLTSTIVTL (SEQ ID NO: 351) variant 1NM_207585_2 T031 IFNGR1 SLWIPVVAALLLFLVLSLVFI (SEQ ID NO: 352)NM_000416_2 T032 IFNGR2 transcriptVILISVGTFSLLSVLAGACFF (SEQ ID NO: 353) variant 1 NM_001329128_1 T033IFNLR1 FLVLPSLLILLLVIAAGGVIW (SEQ ID NO: 354) NM_170743_3 T034IL1R1 transcript HMIGICVTLTVIIVCSVFIYKIF (SEQ ID NO: 355) variant 2NM_001288706_1 T035 IL1RAP transcriptVLLVVILIVVYHVYWLEMVLF (SEQ ID NO: 356) variant 1 NM_002182_3 T036IL1RL1 transcript IYCIIAVCSVFLMLINVLVII (SEQ ID NO: 357) variant 1NM_016232.4 T037 IL1RL2 AYLIGGLIALVAVAVSVVYIY (SEQ ID NO: 358)NM_003854.2 T038 IL2RA transcript VAVAGCVFLLISVLLLSGL (SEQ ID NO: 359)variant 1 NM_000417_2 T039 IL2RB transcriptIPWLGHLLVGLSGAFGFIILVYLLI (SEQ ID NO: 360) variant 1 NM_000878_4 T040IL2RG VVISVGSMGLIISLLCVYFWL (SEQ ID NO: 361) NM_000206_2 T041IL3RA transcript TSLLIALGTLLALVCVFVIC (SEQ ID NO: 362) variant 1 and 2NM_002183_3 T042 IL4R transcriptLLLGVSVSCIVILAVCLLCYVSIT (SEQ ID NO: 363) variant 1 NM_000418_3 T043IL5RA transcript FVIVIMATICFILLILSLIC (SEQ ID NO: 364) variant 1NM_000564_4 T044 IL6R transcript TFLVAGGSLAFGTLLCIAIVL (SEQ ID NO: 365)variant 1 NM_000565_3 T045 IL6ST transcriptAIVVPVCLAFLLTTLLGVLFCF (SEQ ID NO: 366) variant 1 and 3 NM_002184_3 T046IL7RA ILLTISILSFFSVALLVILACVL (SEQ ID NO: 367) NM_002185_3 T047IL7RA Ins PPCL ILLPPCLTISILSFFSVALLVILACVL (SEQ ID NO: 368)(interleukin 7 receptor) T048 IL9R transcriptGNTLVAVSIFLLLTGPTYLLF (SEQ ID NO: 369) variant 1 NM_002186_2 T049IL10RA transcript VIIFFAFVLLLSGALAYCLAL (SEQ ID NO: 370) variant 1NM_001558_3 T050 IL10RB WMVAVILMASVFMVCLALLGCF (SEQ ID NO: 371)NM_000628_4 T051 IL11RA SLGILSFLGLVAGALALGLWL (SEQ ID NO: 372)NM_001142784_2 T052 IL12RB1 transcriptWLIFFASLGSFLSILLVGVLGYLGL (SEQ ID NO: 373) variant 1 and 4 NM_005535_2T053 IL12RB2 transcript WMAFVAPSICIAIIMVGIFST (SEQ ID NO: 374)variant 1 and 3 NM_001559_2 T054 IL13RA1LYITMLLIVPVIVAGAIIVLLLYL (SEQ ID NO: 375) NM_001560_2 T055 IL13RA2FWLPFGFILILVIFVTGLLL (SEQ ID NO: 376) NM_000640_2 T056 IL15RA transcriptVAISTSTVLLCGLSAVSLLACYL (SEQ ID NO: 377) variant 4 NM_001256765_1 T057IL17RA VYWFITGISILLVGSVILLIV (SEQ ID NO: 378) NM_014339_6 T058 IL17RBLLLLSLLVATWVLVAGIYLMW (SEQ ID NO: 379) NM_018725_3 T059IL17RC transcript WALVWLACLLFAAALSLILLL (SEQ ID NO: 380) variant 1NM_153460_3 T060 IL17RD transcriptAVAITVPLVVISAFATLFTVM (SEQ ID NO: 381) variant 2 NM_017563_4 T061IL17RE transcript LGLLILALLALLTLLGVVLAL (SEQ ID NO: 382) variant 1NM_153480_1 T062 IL18R1 transcriptGMIIAVLILVAVVCLVTVCVI (SEQ ID NO: 383) variant 1 NM_003855_3 T063IL18RAP GVVLLYILLGTIGTLVAVLAA (SEQ ID NO: 384) NM_003853_3 T064IL20RA transcript IIFWYVLPISITVFLFSVMGY (SEQ ID NO: 385) variant 1NM_014432_3 T065 IL20RB VLALFAFVGFMLILVVVPLFV (SEQ ID NO: 386)NM_144717_3 T066 IL21R transcript GWNPHLLLLLLLVIVFIPAFW (SEQ ID NO: 387)variant 2 NM_181078_2 T067 IL22RA1YSFSGAFLFSMGFLVAVLCYL (SEQ ID NO: 388) NM_021258_3 T068 IL23RLLLGMIVFAVMLSILSLIGIF (SEQ ID NO: 389) NM_144701_2 T069 IL27RAVLPGILFLWGLFLLGCGLSLA (SEQ ID NO: 390) NM_004843_3 T070 IL27RAVLPGILCLWGLFLLGCGLSLA (SEQ ID NO: 391) NM_004843_3 T071IL31RA transcript IILITSLIGGGLLILIILTVAYGL (SEQ ID NO: 392) variant 1NM_139017_5 T072 LEPR transcriptAGLYVIVPVIISSSILLLGTLLI (SEQ ID NO: 393) variant 1 NM_002303_5 T073 LIFRVGLIIAILIPVAVAVIVGVVTSILC (SEQ ID NO: 394) NM_001127671_1 T074 MPLISLVTALHLVLGLSAVLGLLLL (SEQ ID NO: 395) NM_005373_2 T075 MPLISLVTALHLVLGLNAVLGLLLL (SEQ ID NO: 396) NM_005373_2 T076 OSMR transcriptLIHILLPMVFCVLLIMVMCYL (SEQ ID NO: 397) variant 4 NM_001323505_1 T077PRLR transcript TTVWISVAVLSAVICLIIVWAVAL (SEQ ID NO: 398) variant 1NM_000949_6 T078 TNFRSF4 VAAILGLGLVLGLLGPLAILL (SEQ ID NO: 399)NM_003327_3 T079 TNFRSF8 PVLDAGPVLFWVILVLVVVVGSSAFLLC (SEQ ID NO: 400)transcript variant 1 NM_001243_4 T080 TNFRSF9IISFFLALTSTALLFLLFFLTLRFSVV (SEQ ID NO: 401) NM_001561_5 T081 TNFRSF14WWFLSGSLVIVIVCSTVGLII (SEQ ID NO: 402) transcript variant 1 NM_003820_3T082 TNFRSF18 LGWLTVVLLAVAACVLLLTSA (SEQ ID NO: 403) transcript variant1 NM_004195_2 S036 CD2 transcriptTKRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRPPPPGHRVQvariant 1HQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN (SEQ ID NO: 404)NM_001328609_1 S037 CD3D transcriptGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK (SEQ ID NO: 405) variant 1NM_000732_4 S038 CD3EKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO: 406)NM_000733_3 S039 CD3GGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO: 407)NM_000073_2 S042 CD4 transcriptCVRCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPI (SEQ ID NO: 408)variant 1 and 2 NM_000616_4 S043 CD8A transcriptLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV (SEQ ID NO: 409) variant 1 NM_001768_6S044 CD8B transcriptRRRRARLRFMKQPQGEGISGTFVPQCLHGYYSNTTTSQKLLNPWILKT (SEQ ID NO: 410)variant 2 NM_172213_3 S045 CD8B transcriptRRRRARLRFMKQLRLHPLEKCSRMDY (SEQ ID NO: 411) variant 3 NM_172101_3 S046CD8B transcript RRRRARLRFMKQFYK (SEQ ID NO: 412) variant 5 NM_004931_4S047 CD27QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 413)NM_001242_4 S048 mutated Delta LckRSKRSRLLHSDYMNMTPRRPGPTRKHYQAYAAARDFAAYRS (SEQ ID NO: 414)CD28 transcript variant 1 NM_006139_3 S049 CD28 transcriptRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 415) variant 1NM_006139_3 S050 CD40 transcriptKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ (SEQ IDvariant 1 and 6 NO: 416) NM_001250_5 S051 CD40 transcriptSESSEKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ (SEQ IDvariant 5 NO: 417) NM_001322421_1 S052 CD79A transcriptRKRWQNEKLGLDAGDEYEDENLYEGLNLDDCSMYEDISRGLQGTYQDVGSLNIGDVQLEKP (SEQ IDvariant 1 NO: 418) NM_001783_3 S053 CD79B transcriptLDKDDSKAGMEEDHTYEGLDIDQTATYEDIVTLRTGEVKWSVGEHPGQE (SEQ ID NO: 419)variant 3 NM_001039933_2 S054 CRLF2 transcriptKLWRVKKFLIPSVPDPKSIFPGLFEIHQGNFQEWITDTQNVAHLHKMAGAEQESGPEEPLVVQLAKTEAESPRvariant 1MLDPQTEEKEASGGSLQLPHQPLQGGDVVTIGGFTFVMNDRSYVAL (SEQ ID NO: 420)NM_022148_3 S057 CFS2RBRFCGIYGYRLRRKWEEKIPNPSKSHLFQNGSAELWPPGSMSAFTSGSPPHQGPWGSRFPELEGVFPVGFGDSNM_000395_2EVSPLTIEDPKHVCDPPSGPDTTPAASDLPTEQPPSPQPGPPAASHTPEKQASSFDFNGPYLGPPHSRSLPDILGQPEPPQEGGSQKSPPPGSLEYLCLPAGGQVQLVPLAQAMGPGQAVEVERRPSQGAAGSPSLESGGGPAPPALGPRVGGQDQKDSPVAIPMSSGDTEDPGVASGYVSSADLVFTPNSGASSVSLVPSLGLPSDQTPSLCPGLASGPPGAPGPVKSGFEGYVELPPIEGRSPRSPRNNPVPPEAKSPVLNPGERPADVSPTSPQPEGLLVLQQVGDYCFLPGLGPGPLSLRSKPSSPGPGPEIKNLDQAFQVKKPPGQAVPQVPVIQLFKALKQQDYLSLPPWEVNKPGEVC (SEQ ID NO: 421) S058 CSF2RA transcriptKRFLRIQRLFPPVPQIKDKLNDNHEVEDEIIWEEFTPEEGKGYREEVLTVKEIT (SEQ ID NO: 422)variant 7 and 8 NM_001161529_1 S059 CSF2RA transcriptKRFLRIQRLFPPVPQIKDKLNDNHEVEDEMGPQRHHRCGWNLYPTPGPSPGSGSSPRLGSESSL (SEQ IDvariant 9 NO: 423) NM_001161531_1 S062 CSF3R transcriptSPNRKNPLWPSVPDPAHSSLGSWVPTIMEEDAFQLPGLGTPPITKLTVLEEDEKKPVPWESHNSSETCGLPTLvariant 1VQTYVLQGDPRAVSTQPQSQSGTSDQVLYGQLLGSPTSPGPGHYLRCDSTQPLLAGLTPSPKSYENLWFQASNM_000760_3 PLGTLVTPAPSQEDDCVFGPLLNFPLLQGIRVHGMEALGSF (SEQ ID NO: 424)S063 CSF3R transcriptSPNRKNPLWPSVPDPAHSSLGSWVPTIMEELPGPRQGQWLGQTSEMSRALTPHPCVQDAFQLPGLGTPPIvariant 3TKLTVLEEDEKKPVPWESHNSSETCGLPTLVQTYVLQGDPRAVSTQPQSQSGTSDQVLYGQLLGSPTSPGPGNM_156039_3HYLRCDSTQPLLAGLTPSPKSYENLWFQASPLGTLVTPAPSQEDDCVFGPLLNFPLLQGIRVHGMEALGSF(SEQ ID NO: 425) S064 CSF3R transcriptSPNRKNPLWPSVPDPAHSSLGSWVPTIMEEDAFQLPGLGTPPITKLTVLEEDEKKPVPWESHNSSETCGLPTLvariant 4VQTYVLQGDPRAVSTQPQSQSGTSDQAGPPRRSAYFKDQIMLHPAPPNGLLCLFPITSVL (SEQ ID NO: 426)NM_172313_2 S069 EPOR transcriptHRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTEDPPASLEVLSERCWGTMQAvariant 1VEPGTDDEGPLLEPVGSEHAQDTYLVLDKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASSCSSALASKPSPENM_000121_3GASAASFEYTILDPSSQLLRPWTLCPELPPTPPHLKYLYLVVSDSGISTDYSSGDSQGAQGGLSDGPYSNPYENSLIPAAEPLPPSYVACS (SEQ ID NO: 427) S072 EPOR transcriptHRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTEDPPASLEVLSERCWGTMQAvariant 1VEPGTDDEGPLLEPVGSEHAQDTYLVLDKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASSCSSALASKPSPENM_000121_3GASAASFEYTILDPSSQLLRPWTLCPELPPTPPHLKFLFLVVSDSGISTDYSSGDSQGAQGGLSDGPYSNPYENSLIPAAEPLPPSYVACS (SEQ ID NO: 428) S074 FCER1GRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ (SEQ ID NO: 429) NM_004106_1S075 FCGR2CCRKKRISANSTDPVKAAQFEPPGRQMIAIRKRQPEETNNDYETADGGYMTLNPRAPTDDDKNIYLTLPPNDHNM_201563_5 VNSNN (SEQ ID NO: 430) S076 FCGRA2 transcriptCRKKRISANSTDPVKAAQFEPPGRQMIAIRKRQLEETNNDYETADGGYMTLNPRAPTDDDKNIYLTLPPNDHvariant 1 VNSNN (SEQ ID NO: 431) NM_001136219_1 S077 GHR transcriptKQQRIKMLILPPVPVPKIKGIDPDLLKEGKLEEVNTILAIHDSYKPEFHSDDSWVEFIELDIDEPDEKTEESDTDRLvariant 1LSSDHEKSHSNLGVKDGDSGRTSCCEPDILETDFNANDIHEGTSEVAQPQRLKGEADLLCLDQKNQNNSPYHNM_000163_4DACPATQQPSVIQAEKNKPQPLPTEGAESTHQAAHIQLSNPSSLSNIDFYAQVSDITPAGSVVLSPGQKNKAGMSQCDMHPEMVSLCQENFLMDNAYFCEADAKKCIPVAPHIKVESHIQPSLNQEDIYITTESLTTAAGRPGTGEHVPGSEMPVPDYTSIHIVQSPQGLILNATALPLPDKEFLSSCGYVSTDQLNKIMP (SEQ ID NO: 432)S080 ICOS CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL (SEQ ID NO: 433)NM_012092.3 S081 IFNAR1KVFLRCINYVFFPSLKPSSSIDEYFSEQPLKNLLLSTSEEQIEKCFIIENISTIATVEETNQTDEDHKKYSSQTSQDSNM_000629_2 GNYSNEDESESKTSEELQQDFV (SEQ ID NO: 434) S082IFNAR2 transcriptKWIGYICLRNSLPKVLNFHNFLAWPFPNLPPLEAMDMVEVIYINRKKKVWDYNYDDESDSDTEAAPRTSGGvariant 1GYTMHGLTVRPLGQASATSTESQLIDPESEEEPDLPEVDVELPTMPKDSPQQLELLSGPCERRKSPLQDPFPEENM_207585_2DYSSTEGSGGRITFNVDLNSVFLRVLDDEDSDDLEAPLMLSSHLEEMVDPEDPDNVQSNHLLASGEGTQPTFPSPSSEGLWSEDAPSDQSDTSESDVDLGDGYIMR (SEQ ID NO: 435) S083IFNAR2 transcriptKWIGYICLRNSLPKVLRQGLAKGWNAVAIHRCSHNALQSETPELKQSSCLSFPSSWDYKRASLCPSD (SEQ IDvariant 2 NO: 436) NM_000874_4 S084 IFNGR1CFYIKKINPLKEKSIILPKSLISVVRSATLETKPESKYVSLITSYQPFSLEKEVVCEEPLSPATVPGMHTEDNPGKVENM_000416_2HTEELSSITEVVTTEENIPDVVPGSHLTPIERESSSPLSSNQSEPGSIALNSYHSRNCSESDHSRNGFDTDSSCLESHSSLSDSEFPPNNKGEIKTEGQELITVIKAPTSFGYDKPHVLVDLLVDDSGKESLIGYRPTEDSKEFS (SEQ IDNO: 437) S085 IFNGR2 transcriptLVLKYRGLIKYWFHTPPSIPLQIEEYLKDPTQPILEALDKDSSPKDDVWDSVSIISFPEKEQEDVLQTL (SEQ IDvariant 1 NO: 438) NM_001329128_1 S086 IFNLR1KTLMGNPWFQRAKMPRALDFSGHTHPVATFQPSRPESVNDLFLCPQKELTRGVRPTPRVRAPATQQTRWKNM_170743_3KDLAEDEEEEDEEDTEDGVSFQPYIEPPSFLGQEHQAPGHSEAGGVDSGRPRAPLVPSEGSSAWDSSDRSWASTVDSSWDRAGSSGYLAEKGPGQGPGGDGHQESLPPPEFSKDSGFLEELPEDNLSSWATWGTLPPEPNLVPGGPPVSLQTLTFCWESSPEEEEEARESEIEDSDAGSWGAESTQRTEDRGRTLGHYMAR (SEQ ID NO: 439)S087 IFNLR1 transcriptKTLMGNPWFQRAKMPRALELTRGVRPTPRVRAPATQQTRWKKDLAEDEEEEDEEDTEDGVSFQPYIEPPSFvariant 2LGQEHQAPGHSEAGGVDSGRPRAPLVPSEGSSAWDSSDRSWASTVDSSWDRAGSSGYLAEKGPGQGPGGNM_173064_2DGHQESLPPPEFSKDSGFLEELPEDNLSSWATWGTLPPEPNLVPGGPPVSLQTLTFCWESSPEEEEEARESEIEDSDAGSWGAESTQRTEDRGRTLGHYMAR (SEQ ID NO: 440) S098 IL1R1 transcriptKIDIVLWYRDSCYDFLPIKVLPEVLEKQCGYKLFIYGRDDYVGEDIVEVINENVKKSRRLIIILVRETSGFSWLGGSvariant 2SEEQIAMYNALVQDGIKVVLLELEKIQDYEKMPESIKFIKQKHGAIRWSGDFTQGPQSAKTRFWKNVRYHMPNM_001288706_1 VQRRSPSSKHQLLSPATKEKLQREAHVPLG (SEQ ID NO: 441) S099IL1R1 transcriptKIDIVLWYRDSCYDFLPIKASDGKTYDAYILYPKTVGEGSTSDCDIFVFKVLPEVLEKQCGYKLFIYGRDDYVGEDvariant 3IVEVINENVKKSRRLIIILVRETSGFSWLGGSSEEQIAMYNALVQDGIKVVLLELEKIQDYEKMPESIKFIKQKHGNM_001320978_1AIRWSGDFTQGPQSAKTRFWKNVRYHMPVQRRSPSSKHQLLSPATKEKLQREAHVPLG (SEQ ID NO: 442)S100 IL1RAP transcriptYRAHFGTDETILDGKEYDIYVSYARNAEEEEFVLLTLRGVLENEFGYKLCIFDRDSLPGGIVTDETLSFIQKSRRLLvariant 1VVLSPNYVLQGTQALLELKAGLENMASRGNINVILVQYKAVKETKVKELKRAKTVLTVIKWKGEKSKYPQGRFNM_002182_3 WKQLQVAMPVKKSPRRSSSDEQGLSYSSLKNV (SEQ ID NO: 443) S101IL1RAP transcriptYRAHFGTDETILDGKEYDIYVSYARNAEEEEFVLLTLRGVLENEFGYKLCIFDRDSLPGGNTVEAVFDFIQRSRRvariant 6MIVVLSPDYVTEKSISMLEFKLGVMCQNSIATKLIVVEYRPLEHPHPGILQLKESVSFVSWKGEKSKHSGSKFWNM_001167931_1KALRLALPLRSLSASSGWNESCSSQSDISLDHVQRRRSRLKEPPELQSSERAAGSPPAPGTMSKHRGKSSATCRCCVTYCEGENHLRNKSRAEIHNQPQWETHLCKPVPQESETQWIQNGTRLEPPAPQISALALHHFTDLSNNNDFYIL (SEQ ID NO: 444) S102 IL1RL1 transcriptLKMFWIEATLLWRDIAKPYKTRNDGKLYDAYVVYPRNYKSSTDGASRVEHFVHQILPDVLENKCGYTLCIYGRvariant 1DMLPGEDVVTAVETNIRKSRRHIFILTPQITHNKEFAYEQEVALHCALIQNDAKVILIEMEALSELDMLQAEALNM_016232.4QDSLQHLMKVQGTIKWREDHIANKRSLNSKFWKHVRYQMPVPSKIPRKASSLTPLAAQKQ (SEQ IDNO: 445) S103 IL1RL2NIFKIDIVLWYRSAFHSTETIVDGKLYDAYVLYPKPHKESQRHAVDALVLNILPEVLERQCGYKLFIFGRDEFPGNM_003854.2QAVANVIDENVKLCRRLIVIVVPESLGFGLLKNLSEEQIAVYSALIQDGMKVILIELEKIEDYTVMPESIQYIKQKHGAIRWHGDFTEQSQCMKTKFWKTVRYHMPPRRCRPFPPVQLLQHTPCYRTAGPELGSRRKKCTLTTG (SEQID NO: 446) S104 IL2RA transcript TWQRRQRKSRRTI (SEQ ID NO: 447)variant 1 NM_000417_2 S105 IL2RB transcriptNCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQvariant 1QDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGNM_000878_4EDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV (SEQ IDNO: 448) S106 IL2RGERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPNM_000206_2 YWAPPCYTLKPET (SEQ ID NO: 449) S109 IL3RA transcriptRRYLVMQRLFPRIPHMKDPIGDSFQNDKLVVWEAGKAGLEECLVTEVQVVQKT (SEQ ID NO: 450)variant 1 and 2 NM_002183_3 S110 IL4R transcriptKIKKEWWDQIPNPARSRLVAIIIQDAQGSQWEKRSRGQEPAKCPHWKNCLTKLLPCFLEHNMKRDEDPHKAvariant 1AKEMPFQGSGKSAWCPVEISKTVLWPESISVVRCVELFEAPVECEEEEEVEEEKGSFCASPESSRDDFQEGRENM_000418_3GIVARLTESLFLDLLGEENGGFCQQDMGESCLLPPSGSTSAHMPWDEFPSAGPKEAPPWGKEQPLHLEPSPPASPTQSPDNLTCTETPLVIAGNPAYRSFSNSLSQSPCPRELGPDPLLARHLEEVEPEMPCVPQLSEPTTVPQPEPETWEQILRRNVLQHGAAAAPVSAPTSGYQEFVHAVEQGGTQASAVVGLGPPGEAGYKAFSSLLASSAVSPEKCGFGASSGEEGYKPFQDLIPGCPGDPAPVPVPLFTFGLDREPPRSPQSSHLPSSSPEHLGLEPGEKVEDMPKPPLPQEQATDPLVDSLGSGIVYSALTCHLCGHLKQCHGQEDGGQTPVMASPCCGCCCGDRSSPPTTPLRAPDPSPGGVPLEASLCPASLAPSGISEKSKSSSSFHPAPGNAQSSSQTPKIVNFVSVGPTYMRVS (SEQ ID NO: 451)S113 IL4R transcriptKIKKEWWDQIPNPARSRLVAIIIQDAQGSQWEKRSRGQEPAKCPHWKNCLTKLLPCFLEHNMKRDEDPHKAvariant 1AKEMPFQGSGKSAWCPVEISKTVLWPESISVVRCVELFEAPVECEEEEEVEEEKGSFCASPESSRDDFQEGRENM_000418_3GIVARLTESLFLDLLGEENGGFCQQDMGESCLLPPSGSTSAHMPWDEFPSAGPKEAPPWGKEQPLHLEPSPPASPTQSPDNLTCTETPLVIAGNPAYRSFSNSLSQSPCPRELGPDPLLARHLEEVEPEMPCVPQLSEPTTVPQPEPETWEQILRRNVLQHGAAAAPVSAPTSGYQEFVHAVEQGGTQASAVVGLGPPGEAGYKAFSSLLASSAVSPEKCGFGASSGEEGYKPFQDLIPGCPGDPAPVPVPLFTFGLDREPPRSPQSSHLPSSSPEHLGLEPGEKVEDMPKPPLPQEQATDPLVDSLGSGIVFSALTCHLCGHLKQCHGQEDGGQTPVMASPCCGCCCGDRSSPPTTPLRAPDPSPGGVPLEASLCPASLAPSGISEKSKSSSSFHPAPGNAQSSSQTPKIVNFVSVGPTYMRVS (SEQ ID NO: 452)S115 IL5RA transcriptKICHLWIKLFPPIPAPKSNIKDLFVTTNYEKAGSSETEIEVICYIEKPGVETLEDSVF (SEQ ID NO: 453)variant 1 NM_000564_4 S116 IL6R transcriptRFKKTWKLRALKEGKTSMHPPYSLGQLVPERPRPTPVLVPLISPPVSPSSLGSDNTSSHNRPDARDPRSPYDISvariant 1 NTDYFFPR (SEQ ID NO: 454) NM_000565_3 S117 IL6ST transcriptNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPEDLKSLDLvariant 1 and 3FKKEKINTEGHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRSESTQPLLDNM_002184_3SEERPEDLQLVDHVDGGDGILPRQQYFKQNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAFGPGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQGGYMPQ (SEQ IDNO: 455) S120 IL7RA Isoform 1WKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESENM_002185.4KQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ (SEQ ID NO: 456) S121IL7RA Isoform 3 WKKRIKPIVWPSLPDHKKTLEHLCKKPRKVSVFGA (SEQ ID NO: 457)(C-term deletion) (interleukin 7 receptor) S126 IL9R transcriptKLSPRVKRIFYQNVPSPAMFFQPLYSVHNGNFQTWMGAHGAGVLLSQDCAGTPQGALEPCVQEATALLTCvariant 1GPARPWKSVALEEEQEGPGTRLPGNLSSEDVLPAGCTEWRVQTLAYLPQEDWAPTSLTRPAPPDSEGSRSSSNM_002186_2SSSSSNNNNYCALGCYGGWHLSALPGNTQSSGPIPALACGLSCDHQGLETQQGVAWVLAGHCQRPGLHEDLQGMLLPSVLSKARSWTF (SEQ ID NO: 458) S129 IL10RA transcriptQLYVRRRKKLPSVLLFKKPSPFIFISQRPSPETQDTIHPLDEEAFLKVSPELKNLDLHGSTDSGFGSTKPSLQTEEPvariant 1QFLLPDPHPQADRTLGNREPPVLGDSCSSGSSNSTDSGICLQEPSLSPSTGPTWEQQVGSNSRGQDDSGIDLNM_001558_3VQNSEGRAGDTQGGSALGHHSPPEPEVPGEEDPAAVAFQGYLRQTRCAEEKATKTGCLEEESPLTDGLGPKFGRCLVDEAGLHPPALAKGYLKQDPLEMTLASSGAPTGQWNQPTEEWSLLALSSCSDLGISDWSFAHDLAPLGCVAAPGGLLGSFNSDLVTLPLISSLOSSE (SEQ ID NO: 459) S130 IL10RBALLWCVYKKTKYAFSPRNSLPQHLKEFLGHPHHNTLLFFSFPLSDENDVFDKLSVIAEDSESGKQNPGDSCSLGNM_000628_4 TPPGQGPQS (SEQ ID NO: 460) S135 IL11RARLRRGGKDGSPKPGFLASVIPVDRRPGAPNL (SEQ ID NO: 461) NM_001142784_2 S136IL12RB1 transcriptNRAARHLCPPLPTPCASSAIEFPGGKETWQWINPVDFQEEASLQEALVVEMSWDKGERTEPLEKTELPEGAPvariant 1 and 4 ELALDTELSLEDGDRCKAKM (SEQ ID NO: 462) NM_005535_2 S137IL12RB1 transcriptNRAARHLCPPLPTPCASSAIEFPGGKETWQWINPVDFQEEASLQEALVVEMSWDKGERTEPLEKTELPEGAPvariant 3 ELALDTELSLEDGDRCDR (SEQ ID NO: 463) NM_001290023_1 S138IL12RB2 transcriptHYFQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHQVTPVFvariant 1 and 3RHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVNM_001559_2LPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML(SEQ ID NO: 464) S141 IL13RA1KRLKIIIFPPIPDPGKIFKEMFGDQNDDTLHWKKYDIYEKQTKEETDSVVLIENLKKASQ (SEQ ID NO: 465)NM_001560_2 S142 IL13RA2 RKPNTYPKMIPEFFCDT (SEQ ID NO: 466) NM_000640_2S143 IL15RA transcriptKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO: 467) variant 4NM_001256765_1 S144 IL17RACMTWRLAGPGSEKYSDDTKYTDGLPAADLIPPPLKPRKVWIIYSADHPLYVDVVLKFAQFLLTACGTEVALDLLNM_014339_6EEQAISEAGVMTWVGRQKQEMVESNSKIIVLCSRGTRAKWQALLGRGAPVRLRCDHGKPVGDLFTAAMNMILPDFKRPACFGTYVVCYFSEVSCDGDVPDLFGAAPRYPLMDRFEEVYFRIQDLEMFQPGRMHRVGELSGDNYLRSPGGRQLRAALDRFRDWQVRCPDWFECENLYSADDQDAPSLDEEVFEEPLLPPGTGIVKRAPLVREPGSQACLAIDPLVGEEGGAAVAKLEPHLQPRGQPAPQPLHTLVLAAEEGALVAAVEPGPLADGAAVRLALAGEGEACPLLGSPGAGRNSVLFLPVDPEDSPLGSSTPMASPDLLPEDVREHLEGLMLSLFEQSLSCQAQGGCSRPAMVLTDPHTPYEEEQRQSVQSDQGYISRSSPQPPEGLTEMEEEEEEEQDPGKPALPLSPEDLESLRSLQRQLLFRQLQKNSGWDTMGSESEGPSA (SEQ ID NO: 468) S145 IL17RBRHERIKKTSFSTTTLLPPIKVLVVYPSEICFHHTICYFTEFLQNHCRSEVILEKWQKKKIAEMGPVQWLATQKKANM_018725_3ADKVVFLLSNDVNSVCDGTCGKSEGSPSENSQDLFPLAFNLFCSDLRSQIHLHKYVVVYFREIDTKDDYNALSVCPKYHLMKDATAFCAELLHVKQQVSAGKRSQACHDGCCSL (SEQ ID NO: 469) S146IL17RC transcriptKKDHAKGWLRLLKQDVRSGAAARGRAALLLYSADDSGFERLVGALASALCQLPLRVAVDLWSRRELSAQGPvariant 1VAWFHAQRRQTLQEGGVVVLLFSPGAVALCSEWLQDGVSGPGAHGPHDAFRASLSCVLPDFLQGRAPGSYNM_153460_3VGACFDRLLHPDAVPALFRTVPVFTLPSQLPDFLGALQQPRAPRSGRLQERAEQVSRALQPALDSYFHPPGTPAPGRGVGPGAGPGAGDGT (SEQ ID NO: 470) S147 IL17RC transcriptKKDHAKAAARGRAALLLYSADDSGFERLVGALASALCQLPLRVAVDLWSRRELSAQGPVAWFHAQRRQTLQvariant 4EGGVVVLLFSPGAVALCSEWLQDGVSGPGAHGPHDAFRASLSCVLPDFLQGRAPGSYVGACFDRLLHPDAVNM_001203263_1PALFRTVPVFTLPSQLPDFLGALQQPRAPRSGRLQERAEQVSRALQPALDSYFHPPGTPAPGRGVGPGAGPGAGDGT (SEQ ID NO: 471) S148 IL17RD transcriptCRKKQQENIYSHLDEESSESSTYTAALPRERLRPRPKVFLCYSSKDGQNHMNVVQCFAYFLQDFCGCEVALDLvariant 2WEDFSLCREGQREWVIQKIHESQFIIVVCSKGMKYFVDKKNYKHKGGGRGSGKGELFLVAVSAIAEKLRQAKNM_017563_4QSSSAALSKFIAVYFDYSCEGDVPGILDLSTKYRLMDNLPQLCSHLHSRDHGLQEPGQHTRQGSRRNYFRSKSGRSLYVAICNMHQFIDEEPDWFEKQFVPFHPPPLRYREPVLEKFDSGLVLNDVMCKPGPESDFCLKVEAAVLGATGPADSQHESQHGGLDQDGEARPALDGSAALQPLLHTVKAGSPSDMPRDSGIYDSSVPSSELSLPLMEGLSTDQTETSSLTESVSSSSGLGEEEPPALPSKLLSSGSCKADLGCRSYTDELHAVAPL (SEQ ID NO: 472)S149 IL17RE transcriptTCRRPQSGPGPARPVLLLHAADSEAQRRLVGALAELLRAALGGGRDVIVDLWEGRHVARVGPLPWLWAARvariant 1TRVAREQGTVLLLWSGADLRPVSGPDPRAAPLLALLHAAPRPLLLLAYFSRLCAKGDIPPPLRALPRYRLLRDLPNM_153480_1RLLRALDARPFAEATSWGRLGARQRRQSRLELCSRLEREAARLADLG (SEQ ID NO: 473) S154IL18R1 transcriptYRVDLVLFYRHLTRRDETLTDGKTYDAFVSYLKECRPENGEEHTFAVEILPRVLEKHFGYKLCIFERDVVPGGAVvariant 1VDEIHSLIEKSRRLIIVLSKSYMSNEVRYELESGLHEALVERKIKIILIEFTPVTDFTFLPQSLKLLKSHRVLKWKADNM_003855_3 KSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVLPVLSES (SEQ ID NO: 474) S155IL18RAPSALLYRHWIEIVLLYRTYQSKDQTLGDKKDFDAFVSYAKWSSFPSEATSSLSEEHLALSLFPDVLENKYGYSLCLLNM_003853_3ERDVAPGGVYAEDIVSIIKRSRRGIFILSPNYVNGPSIFELQAAVNLALDDQTLKLILIKFCYFQEPESLPHLVKKALRVLPTVTWRGLKSVPPNSRFWAKMRYHMPVKNSQGFTWNQLRITSRIFQWKGLSRTETTGRSSQPKEW(SEQ ID NO: 475) S156 IL20RA transcriptSIYRYIHVGKEKHPANLILIYGNEFDKRFFVPAEKIVINFITLNISDDSKISHQDMSLLGKSSDVSSLNDPQPSGNLvariant 1RPPQEEEEVKHLGYASHLMEIFCDSEENTEGTSLTQQESLSRTIPPDKTVIEYEYDVRTTDICAGPEEQELSLQEENM_014432_3VSTQGTLLESQAALAVLGPQTLQYSYTPQLQDLDPLAQEHTDSEEGPEEEPSTTLVDWDPQTGRLCIPSLSSFDQDSEGCEPSEGDGLGEEGLLSRLYEEPAPDRPPGENETYLMQFMEEWGLYVQMEN (SEQ ID NO: 476)S157 IL20RBWKMGRLLQYSCCPVVVLPDTLKITNSPQKLISCRREEVDACATAVMSPEELLRAWIS (SEQ ID NO: 477)NM_144717_3 S158 IL21R transcriptSLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSSLELGPWSPEVPSTLEVYSCHPPRSPvariant 2AKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCTWPCSCEDDNM_181078_2GYPALDLDAGLEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQAS(SEQ ID NO: 478) S161 IL22RA1SYRYVTKPPAPPNSLNVQRVLTFQPLRFIQEHVLIPVFDLSGPSSLAQPVQYSQIRVSGPREPAGAPQRHSLSEINM_021258_3TYLGQPDISILQPSNVPPPQILSPLSYAPNAAPEVGPPSYAPQVTPEAQFPFYAPQAISKVQPSSYAPQATPDSWPPSYGVCMEGSGKDSPTGTLSSPKHLRPKGQLQKEPPAGSCMLGGLSLQEVTSLAMEESQEAKSLHQPLGICTDRTSDPNVLHSGEEGTPQYLKGQLPLLSSVQIEGHPMSLPLQPPSRPCSPSDQGPSPWGLLESLVCPKDEAKSPAPETSDLEQPTELDSLFRGLALTVQWES (SEQ ID NO: 479) S165 IL23RNRSFRTGIKRRILLLIPKWLYEDIPNMKNSNVVKMLQENSELMNNNSSEQVLYVDPMITEIKEIFIPEHKPTDYKNM_144701_2KENTGPLETRDYPQNSLFDNTTVVYIPDLNTGYKPQISNFLPEGSHLSNNNEITSLTLKPPVDSLDSGNNPRLQKHPNFAFSVSSVNSLSNTIFLGELSLILNQGECSSPDIQNSVEEETTMLLENDSPSETIPEQTLLPDEFVSCLGIVNEELPSINTYFPQNILESHENRISLLEK (SEQ ID NO: 480) S168 IL27RATSGRCYHLRHKVLPRWVWEKVPDPANSSSGQPHMEQVPEAQPLGDLPILEVEEMEPPPVMESSQPAQATANM_004843_3 PLDSGYEKHFLPTPEELGLLGPPRPQVLA (SEQ ID NO: 481) S169 IL27RATSWVWEKVPDPANSSSGQPHMEQVPEAQPLGDLPILEVEEMEPPPVMESSQPAQATAPLDSGYEKHFLPTNM_004843_3 PEELGLLGPPRPQVLA (SEQ ID NO: 482) S170 IL31RA transcriptKKPNKLTHLCWPTVPNPAESSIATWHGDDFKDKLNLKESDDSVNTEDRILKPCSTPSDKLVIDKLVVNFGNVLvariant 1QEIFTDEARTGQENNLGGEKNGYVTCPFRPDCPLGKSFEELPVSPEIPPRKSQYLRSRMPEGTRPEAKEQLLFSNM_139017_5 GQSLVPDHLCEEGAPNPYLKNSVTAREFLVSEKLPEHTKGEV (SEQ ID NO: 483)S171 IL31RA transcriptKKPNKLTHLCWPTVPNPAESSIATWHGDDFKDKLNLKESDDSVNTEDRILKPCSTPSDKLVIDKLVVNFGNVLvariant 4 QEIFTDEARTGQENNLGGEKNGTRILSSCPTSI (SEQ ID NO: 484)NM_001242638_1 S174 LEPR transcriptSHQRMKKLFWEDVPNPKNCSWAQGLNFQKPETFEHLFIKHTASVTCGPLLLEPETISEDISVDTSWKNKDEMvariant 1MPTTVVSLLSTTDLEKGSVCISDQFNSVNFSEAEGTEVTYEDESQRQPFVKYATLISNSKPSETGEEQGLINSSVNM_002303_5TKCFSSKNSPLKDSFSNSSWEIEAQAFFILSDQHPNIISPHLTFSEGLDELLKLEGNFPEENNDKKSIYYLGVTSIKKRESGVLLTDKSRVSCPFPAPCLFTDIRVLQDSCSHFVENNINLGTSSKKTFASYMPQFQTCSTQTHKIMENKMCDLTV (SEQ ID NO: 485) S175 LEPR transcriptSHQRMKKLFWEDVPNPKNCSWAQGLNFQKMLEGSMFVKSHHHSLISSTQGHKHCGRPQGPLHRKTRDLCvariant 2 SLVYLLTLPPLLSYDPAKSPSVRNTQE (SEQ ID NO: 486) NM_001003680_3S176 LEPR transcript SHQRMKKLFWEDVPNPKNCSWAQGLNFQKRTDIL (SEQ ID NO: 487)variant 3 NM_001003679_3 S177 LEPR transcriptSHQRMKKLFWEDVPNPKNCSWAQGLNFQKKMPGTKELLGGGWLT (SEQ ID NO: 488) variant 5NM_001198688_1 S180 LIFRYRKREWIKETFYPDIPNPENCKALQFQKSVCEGSSALKTLEMNPCTPNNVEVLETRSAFPKIEDTEIISPVAERPNM_001127671_1EDRSDAEPENHVVVSYCPPIIEEEIPNPAADEAGGTAQVIYIDVQSMYQPQAKPEEEQENDPVGGAGYKPQMHLPINSTVEDIAAEEDLDKTAGYRPQANVNTWNLVSPDSPRSIDSNSEIVSFGSPCSINSRQFLIPPKDEDSPKSNGGGWSFTNFFQNKPND (SEQ ID NO: 489) S183 LMP1YYHGQRHSDEHHHDDSLPHPQQATDDSGHESDSNSNEGRHHLLVSGAGDGPPLCSQNLGAPGGGPDNGPNC_007605_1QDPDNTDDNGPQDPDNTDDNGPHDPLPQDPDNTDDNGPQDPDNTDDNGPHDPLPHSPSDSAGNDGGPPQLTEEVENKGGDQGPPLMTDGGGGHSHDSGHGGGDPHLPTLLLGSSGSGGDDDDPHGPVQLSYYD(SEQ ID NO: 490) S186 MPLRWQFPAHYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPLPLCSSQANM_005373_2QMDYRRLQPSCLGTMPLSVCPPMAESGSCCTTHIANHSYLPLSYWQQP (SEQ ID NO: 491) S189MYD88 transcriptMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTvariant 1GRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELNM_001172567_1AGITTLDDPLGHMPERFDAFICYCPSDIQFVQEMIRQLEQTNYRLKLCVSDRDVLPGTCVWSIASELIEKRLARRPRGGCRRMVVVVSDDYLQSKECDFQTKFALSLSPGAHQKRLIPIKYKAMKKEFPSILRFITVCDYTNPCTKSWFWTRLAKALSLP (SEQ ID NO: 492) S190 MYD88 transcriptMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTvariant 2GRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELNM_002468_4AGITTLDDPLGHMPERFDAFICYCPSDIQFVQEMIRQLEQTNYRLKLCVSDRDVLPGTCVWSIASELIEKRCRRMVVVVSDDYLQSKECDFQTKFALSLSPGAHQKRLIPIKYKAMKKEFPSILRFITVCDYTNPCTKSWFWTRLAKALSLP (SEQ ID NO: 493) S191 MYD88 transcriptMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTvariant 3GRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIGHMPERFDAFICYCPSDIQFVQEMIRQLEQTNYRLNM_001172568_1KLCVSDRDVLPGTCVWSIASELIEKRCRRMVVVVSDDYLQSKECDFQTKFALSLSPGAHQKRLIPIKYKAMKKEFPSILRFITVCDYTNPCTKSWFWTRLAKALSLP (SEQ ID NO: 494) S192 MYD88 transcriptMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTvariant 4GRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELNM_001172569_1AGITTLDDPLGAAGWWWLSLMITCRARNVTSRPNLHSASLOVPIRSD (SEQ ID NO: 495) S193MYD88 transcriptMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTvariant 5GRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIGAAGWWWLSLMITCRARNVTSRPNLHSASLQVPINM_001172566_1 RSD (SEQ ID NO: 496) S194 MYD88 transcriptMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTvariant 1GRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELNM_001172567_1 AGITTLDDPLGHMPERFDAFICYCPSDI (SEQ ID NO: 497) S195MYD88 transcriptMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTvariant 3GRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIGHMPERFDAFICYCPSDI (SEQ ID NO: 498)NM_001172568_1 S196 MYD88 transcriptMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTvariant 1GRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELNM_001172567_1AGITTLDDPLGHMPERFDAFICYCPSDIQFVQEMIRQLEQTNYRLKLCVSDRDVLPGTCVWSIASELIEKRLARRPRGGCRRMVVVVSDDYLQSKECDFQTKFALSLSPGAHQKRPIPIKYKAMKKEFPSILRFITVCDYTNPCTKSWFWTRLAKALSLP (SEQ ID NO: 499) S197 MYD88 transcriptMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTvariant 2GRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLOVAAVDSSVPRTAELNM_002468_4AGITTLDDPLGHMPERFDAFICYCPSDIQFVQEMIRQLEQTNYRLKLCVSDRDVLPGTCVWSIASELIEKRCRRMVVVVSDDYLQSKECDFQTKFALSLSPGAHQKRPIPIKYKAMKKEFPSILRFITVCDYTNPCTKSWFWTRLAKALSLP (SEQ ID NO: 500) S198 MYD88 transcriptMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTvariant 3GRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIGHMPERFDAFICYCPSDIQFVQEMIRQLEQTNYRLNM_001172568_1KLCVSDRDVLPGTCVWSIASELIEKRCRRMVVVVSDDYLQSKECDFQTKFALSLSPGAHQKRPIPIKYKAMKKEFPSILRFITVCDYTNPCTKSWFWTRLAKALSLP (SEQ ID NO: 501) S199 OSMR transcriptKSQWIKETCYPDIPDPYKSSILSLIKFKENPHLIIMNVSDCIPDAIEVVSKPEGTKIQFLGTRKSLTETELTKPNYLvariant 4YLLPTEKNHSGPGPCICFENLTYNQAASDSGSCGHVPVSPKAPSMLGLMTSPENVLKALEKNYMNSLGEIPAGENM_001323505_1TSLNYVSQLASPMFGDKDSLPTNPVEAPHCSEYKMQMAVSLRLALPPPTENSSLSSITLLDPGEHYC (SEQ IDNO: 502) S202 PRLR transcriptKGYSMVTCIFPPVPGPKIKGFDAHLLEKGKSEELLSALGCQDFPPTSDYEDLLVEYLEVDDSEDQHLMSVHSKEvariant 1HPSQGMKPTYLDPDTDSGRGSCDSPSLLSEKCEEPQANPSTFYDPEVIEKPENPETTHTWDPQCISMEGKIPYNM_000949_6FHAGGSKCSTWPLPQPSQHNPRSSYHNITDVCELAVGPAGAPATLLNEAGKDALKSSQTIKSREEGKATQQREVESFHSETDQDTPWLLPQEKTPFGSAKPLDYVEIHKVNKDGALSLLPKQRENSGKPKKPGTPENNKEYAKVSGVMDNNILVLVPDPHAKNVACFEESAKEAPPSLEQNQAEKALANFTATSSKCRLQLGGLDYLDPACFTHSFH(SEQ ID NO: 503) S211 TNFRSF4ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 504) NM_003327_3S212 TNFRSF8HRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGAAYLtranscript variantESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEAD1 NM_001243_4 HTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK (SEQ ID NO: 505)S213 TNFRSF9 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 506)NM_001561_5 S214 TNFRSF14CVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFTGRSPNH (SEQ ID NO: 507)transcript variant 1 NM_003820_3 S215 TNFRSF18QLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV (SEQ ID NO: 508)transcript variant 1 NM_004195_2 S216 TNFRSF18QLGLHIWQLRKTQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV (SEQ ID NO: 509)transcript variant 3_NM_148902_1 X001 LinkerGSGGSEGGGSEGGAATAGSGSGS (SEQ ID NO: 510)

TABLE 7 Library 1A Top Constructs. BlockSequence Norm_D7 Enrich_D35BlockSequence Norm_D7 Enrich_D35 M008-S212-S075 1.42 10.58M008-S039-S212 2.44 8.05 M025-S194-S045 22.50 7.23 M049-S195-S213 2.468.03 M018-S195-S104 2.75 9.75 M049-S045-S142 4.08 7.47 M030-S076-S1042.60 9.49 M007-S109-S076 7.95 6.65 M049-S211-S076 7.09 8.24M043-S087-S038 1.42 8.53 M013-S165-S194 2.96 9.15 M049-S081-S074 2.348.06 M013-S038-S059 14.06 7.15 M008-S045-S126 1.62 8.41 M019-S059-S0430.00 10.85 M008-S044-S039 10.46 6.28 M008-S047-S053 7.27 7.78M037-S180-S157 0.00 9.79 M030-S104-S176 2.31 9.10 M007-S099-S059 17.075.61 M013-S121-S053 19.93 6.41 M024-S197-S037 3.32 7.68 M025-S103-S1412.73 8.90 M036-S104-S154 13.42 5.94 M031-S074-S038 2.71 8.86M042-S135-S120 0.00 9.78 M013-S084-S059 9.91 7.30 M037-S059-S045 3.197.70 M007-S137-S047 13.29 6.87 M024-S038-S213 0.00 9.72 M043-S169-S2132.53 8.88 M009-S051-S216 0.00 9.64 M013-S190-S047 0.00 10.68M049-S116-S043 0.00 9.64 M007-S116-S050 10.42 7.14 M025-S183-S189 0.009.61 M043-S106-S176 6.06 7.82 M019-S083-S050 0.00 9.59 M031-S190-S1413.01 8.62 M007-S053-S189 0.00 9.58 M002-S143-S212 9.08 7.24M049-S054-S216 0.00 9.52 M018-S157-S216 24.51 5.90 M049-S154-S143 0.009.47 M019-S136-S214 2.65 8.68 M002-S170-S043 0.00 9.46 M002-S135-S10411.35 6.85 M025-S049-S168 0.00 9.38 M030-S100-S064 7.68 7.35M049-S086-S045 0.00 9.38 M043-S109-S176 3.71 8.22 M036-S045-S043 0.009.35 M049-S083-S190 25.27 5.71 M043-S104-S213 0.00 9.32 M001-S064-S0361.30 9.21 M031-S192-S047 0.00 9.32 M043-S106-S211 13.93 6.49M043-S044-S136 0.00 9.30 M009-S038-S212 5.54 7.66 M048-S171-S075 0.009.30 M007-S216-S052 0.50 9.75 M008-S050-S104 0.00 9.29 M048-S109-S05416.67 6.19 M007-S051-S109 0.00 9.28 M049-S121-S169 12.36 6.58M002-S130-S037 0.00 9.18 M010-S194-S214 2.92 8.34 M002-S106-S141 0.009.16 M019-S169-S038 2.93 8.32 M018-S053-S051 0.00 9.09 M019-S142-S05314.15 6.38 M018-S216-S104 0.00 9.00 M043-S109-S215 0.00 10.29M008-S050-S048 0.00 9.00 M013-S214-S197 24.02 5.64 M007-S051-S085 0.008.98 M019-S104-S157 0.00 10.28 M031-S212-S042 0.00 8.97 M049-S142-S04316.10 6.18 M012-S170-S135 0.00 8.94 M012-S213-S080 0.00 10.27M024-S211-S141 0.00 8.91 M007-S213-S213 34.94 5.09 M008-S039-S158 0.008.89 M007-S115-S189 1.92 8.70 M012-S049-S072 0.00 8.86 M049-S135-S1427.42 7.17 M007-S171-S175 0.00 8.85 M002-S052-S050 20.15 5.84M024-S142-S053 0.00 8.80 M013-S101-S157 1.82 8.72 M042-S044-S075 0.008.77 M031-S142-S051 6.29 7.34 M036-S136-S104 0.00 8.77 M042-S083-S0375.91 7.38 M001-S104-S142 0.00 8.76 M024-S194-S053 0.00 10.16M036-S191-S135 0.00 8.75 M002-S169-S042 14.27 6.22 M043-S214-S043 0.008.73 M010-S192-S136 16.88 5.99 M037-S175-S077 0.00 8.70 M013-S121-S0399.20 6.75 M013-S039-S169 0.00 8.66 M002-S199-S042 16.48 5.97M007-S115-S046 0.00 8.65 M001-S051-S142 20.08 5.70 M001-S202-S085 0.008.63 M030-S052-S121 27.94 5.19 M049-S175-S202 0.00 8.58 M025-S120-S0394.61 7.55 M030-S047-S142 0.00 8.56 M013-S050-S135 28.40 5.13M019-S126-S116 0.00 8.55 M010-S157-S050 2.61 8.14 M019-S213-S058 0.008.55 M042-S109-S116 2.48 8.18 M002-S051-S037 0.00 8.52 M018-S116-S17623.75 5.35 M019-S177-S116 0.01 8.51 M037-S142-S043 7.79 6.84M030-S136-S104 1.03 8.49 M037-S175-S186 2.73 8.07 M002-S043-S109 0.008.49 M009-S059-S069 0.00 9.97 M025-S136-S198 0.00 8.48 M001-S175-S0483.93 7.65 M037-S043-S216 0.00 8.48 M013-S051-S197 4.74 7.42M018-S053-S169 0.00 8.47 M025-S213-S215 22.18 5.41 M049-S155-S104 0.008.46 M049-S080-S046 2.12 8.30 M007-S199-S135 0.00 8.46 M007-S121-S0501.94 8.37 M018-S175-S058 0.00 8.45 M019-S135-S216 27.12 5.11M048-S191-S142 0.00 8.45 M031-S116-S052 14.89 5.92 M007-S171-S051 0.008.44 M043-S183-S038 1.31 8.70 M037-S137-Z001 0.00 8.44 M025-S080-S10218.66 5.60 M009-S183-S183 0.75 8.44 M007-S158-S084 13.62 6.03M002-S143-S075 0.00 8.43 M009-S042-S047 15.99 5.81 M025-S036-S215 0.008.43 M008-S050-S062 14.05 5.98 M049-S183-S135 0.00 8.43 M043-S195-S04830.26 4.93 M008-S080-S145 0.00 8.41 M037-S044-S058 13.82 6.00M009-S052-S039 0.00 8.39 M030-S212-S045 14.24 5.95 M002-S212-S049 0.008.39 M031-S053-S143 5.48 7.18 M007-S137-S202 0.00 8.39 M019-S168-S2165.39 7.19 M025-S037-S050 0.00 8.39 M013-S047-S046 6.89 6.89M024-S189-S076 0.01 8.38 M007-S042-S215 5.27 7.22 M019-S083-S155 0.008.38 M049-S143-S053 2.99 7.85 M049-S214-S054 0.00 8.38 M037-S076-S1150.00 9.85 M043-S126-S037 0.00 8.38 M043-S081-S098 10.07 6.38M012-S054-S194 0.00 8.38 M025-S142-S048 3.67 7.62 M018-S038-S169 0.958.37

TABLE 8 Library 2B Top Constructs. BlockSequence Norm_D7 Enrich_D28BlockSequence Norm_D7 Enrich_D28 M024-S190-S047 30.96 10.78M018-S199-S083 6.87 6.78 M025-S050-S197 2.95 13.25 M008-S074-S072 7.986.59 M036-S170-S047 0.00 13.79 M043-S053-S039 20.99 5.30 M012-S045-S0481.35 12.24 M018-S115-S213 7.37 6.69 M049-S194-S064 10.08 9.04M002-S069-S051 10.80 6.19 M019-S161-S215 10.44 8.59 M037-S046-S053 18.415.47 M025-S190-S050 25.07 7.20 M049-S138-S191 7.49 6.67 M013-S141-S0452.58 9.78 M010-S103-S197 8.34 6.53 M036-S104-S062 10.56 8.06M030-S198-S043 13.25 5.92 M001-S099-S080 7.86 8.42 M013-S215-S215 15.215.73 M018-S053-S130 4.30 8.84 M019-S130-S135 4.30 7.34 M037-S081-S1424.67 8.67 M001-S190-S047 8.72 6.47 M030-S170-S168 8.60 7.83M024-S176-S046 4.42 7.31 M001-S174-S044 8.59 7.81 M030-S064-S121 7.246.70 M002-S190-S183 9.34 7.63 M049-S064-S100 1.60 8.37 M049-S105-S08412.28 7.22 M008-S176-S074 10.31 6.24 M019-S046-S197 2.45 9.08M002-S051-S102 0.00 9.69 M036-S085-S044 8.23 7.61 M001-S063-S039 0.009.68 M030-S059-S106 1.47 9.50 M007-S050-S039 0.00 9.67 M048-S157-S15410.32 7.30 M030-S141-S045 0.00 9.66 M025-S177-S194 13.51 6.88M018-S197-S137 0.00 9.63 M019-S214-S138 17.44 6.51 M019-S098-S126 0.009.62 M037-S120-S085 8.35 7.47 M037-S141-S101 0.00 9.61 M037-S043-S07412.77 6.90 M008-S202-S191 0.00 9.61 M009-S213-S037 0.00 10.68M036-S144-S103 0.00 9.61 M049-S192-S212 3.93 8.37 M013-S143-S194 0.009.60 M049-S098-S177 13.39 6.82 M019-S039-S069 0.00 9.58 M043-S069-S1770.74 9.86 M036-S126-S103 0.00 9.57 M025-S072-S047 38.55 5.34M018-S190-S048 0.00 9.56 M030-S043-S194 9.21 7.29 M024-S072-S198 0.009.52 M037-S083-S109 2.82 8.70 M036-S175-S064 0.00 9.49 M025-S154-S2160.00 10.63 M010-S130-S170 0.00 9.49 M049-S138-S047 27.13 5.71M018-S130-S080 0.00 9.41 M002-S103-S158 8.72 7.23 M048-S051-S129 0.009.24 M012-S050-S043 3.19 8.42 M042-S109-S103 0.73 8.87 M002-S036-S12119.90 6.05 M036-S174-S195 0.73 8.85 M031-S083-S130 5.28 7.72M009-S101-S115 0.86 8.83 M043-S186-S087 13.75 6.48 M049-S042-S051 0.008.75 M012-S216-S039 9.45 6.97 M013-S190-S099 0.00 8.74 M043-S214-S10415.84 6.24 M036-S156-S135 0.86 8.72 M049-S076-S157 4.79 7.78M025-S050-S043 0.00 8.71 M008-S186-S076 7.37 7.24 M037-S042-S195 0.868.67 M031-S038-S212 3.80 8.00 M002-S174-S049 0.98 8.65 M030-S130-S2163.56 8.04 M009-S049-S038 0.98 8.59 M007-S049-S051 0.00 10.22M010-S190-S050 0.00 8.58 M042-S082-S158 12.40 6.47 M018-S053-S156 1.238.57 M037-S194-S194 10.07 6.75 M024-S084-S157 1.23 8.56 M031-S135-S1836.75 7.23 M007-S189-S069 0.86 8.55 M043-S156-S099 5.77 7.41M030-S197-S130 1.10 8.53 M012-S186-S169 3.81 7.89 M018-S130-S059 0.008.51 M048-S038-S076 10.55 6.59 M042-S155-S195 1.47 8.43 M042-S059-S1068.34 6.82 M010-S052-S038 1.10 8.42 M037-S074-S175 1.47 8.69M042-S087-S142 1.35 8.42 M012-S121-S106 14.24 6.05 M018-S062-S105 0.008.39 M013-S075-S212 17.18 5.74 M010-S099-S202 1.47 8.30 M013-S193-S1685.16 7.28 M030-S059-S213 1.35 8.26 M002-S142-S072 36.59 4.65M019-S176-S084 1.35 8.22 M001-S199-S102 5.77 7.11 M037-S058-S194 1.848.21 M010-S062-S192 14.00 5.95 M002-S171-S080 1.84 8.21 M031-S075-S16911.66 6.19 M002-S130-S196 1.84 8.20 M049-S216-S193 10.55 6.31M036-S183-S136 1.72 8.19 M030-S130-S064 5.27 7.19 M048-S106-S053 1.598.18 M031-S158-S046 7.24 6.79 M036-S045-S086 2.09 8.08 M049-S193-S04416.94 5.67 M049-S129-S044 1.72 8.08 M043-S102-S080 4.91 7.25M008-S146-S154 1.96 8.03 M042-S191-S047 1.47 8.51 M030-S051-S199 1.847.95 M008-S156-S161 19.26 5.47 M043-S051-S211 2.33 7.92 M007-S050-S1105.52 7.10 M024-S190-S171 2.33 7.88 M031-S109-S192 5.27 7.15M010-S177-S196 1.35 7.88 M024-S064-S168 14.48 5.83 M049-S050-S212 2.457.87 M018-S215-S213 20.62 5.35 M008-S143-S050 2.33 7.86 M025-S084-S0398.47 6.54 M002-S039-S104 1.72 7.84 M042-S101-S171 5.89 6.99M019-S138-S075 2.70 7.82 M013-S148-S085 14.36 5.83 M019-S116-S177 2.337.82 M031-S054-S138 10.18 6.29 M013-S076-S197 1.59 7.80 M030-S161-S2136.99 6.77 M024-S042-S216 2.57 7.80 M042-S180-S042 10.43 6.26M007-S202-S102 2.33 7.78 M024-S049-S189 1.23 8.61 M002-S043-S104 2.947.76 M025-S121-S052 5.27 7.12 M030-S215-S064 2.58 7.75 M012-S072-Z0015.15 7.14 M001-S098-S126 1.84 7.74 M048-S104-S180 12.27 6.03M013-S085-S046 2.70 7.73 M048-S045-S045 0.00 9.76 M043-S046-S189 2.827.73 M031-S189-S050 7.61 6.66 M009-S059-S050 2.70 7.70 M037-S109-S1352.70 7.87

TABLE 9 Library 1.1A Top Constructs. BlockSequence Norm_D7 Enrich_D42M007-S120-S214 0.00 16.52 M001-S117-S105 2.25 15.48 M024-S062-S137 0.7714.07 M012-S117-S194 4.25 14.05 M012-S117-S126 0.00 13.58 M001-S117-S1690.84 13.18 M012-S062-S046 2.45 12.77 M012-S117-S058 2.96 12.75M048-S054-S076 5.15 12.73 M036-S080-S195 3.09 12.44 M042-S120-S199 3.0912.29 M030-S080-S197 0.00 12.21 M001-S155-S048 1.29 11.96 M012-S171-S0741.09 11.86 M030-S117-S049 5.21 11.74 M030-S170-S080 6.18 11.63M030-S136-S101 4.38 11.59 M024-S130-S063 8.30 11.51 M024-S211-S154 4.8311.45 M012-S062-S138 0.90 11.06 M030-S102-S191 1.42 10.86 M036-S197-S0760.00 10.82 M030-S190-S047 0.00 10.75 M012-S062-S169 11.00 10.59M012-S037-S157 0.90 10.52 M030-S054-S169 0.97 10.42 M012-S117-S142 4.3110.40 M042-S038-S053 1.54 10.24 M001-S186-S084 3.02 10.17 M024-S130-S1710.00 10.05 M042-S190-S042 3.41 9.93 M036-S143-S194 0.39 9.92M030-S168-S137 7.34 9.81 M012-S062-S136 1.29 9.77 M030-S084-S143 7.539.60 M012-S054-S169 2.70 9.51 M012-S062-S170 14.22 9.34 M030-S062-S1430.45 9.34 M030-S154-S194 3.09 9.28 M012-S064-S063 3.35 9.25M030-S170-S194 7.27 9.19 M030-S170-S168 3.67 9.14 M012-S106-S169 1.359.10 M001-S117-S052 2.64 9.10 M030-S198-S169 1.03 9.04 M030-S214-S0502.32 9.02 M036-S199-S038 1.61 9.01 M018-S044-S177 4.38 8.85M001-S190-S042 0.00 8.79 M036-S175-S059 10.04 8.78

TABLE 10 Library 1.1B Top Constructs. BlockSequence Norm_D7 Enrich_D28M001-S126-S170 2.57 7.68 M012-S106-S169 1.35 7.65 M007-S194-S100 0.007.19 M036-S101-S183 0.00 7.11 M024-S135-S098 0.00 7.08 M024-S038-S0980.00 7.04 M007-S109-S135 0.00 7.02 M024-S191-S038 0.00 6.94M012-S064-S116 0.00 6.93 M009-S100-S074 0.00 6.84 M001-S120-S039 0.006.84 M018-S154-S085 0.00 6.83 M030-S104-S048 0.00 6.79 M030-S168-S1210.00 6.78 M042-S120-S171 1.16 6.75 M036-S176-S195 0.90 6.62M010-S171-S169 0.00 6.53 M030-S175-S120 1.48 6.52 M048-S051-S115 0.846.52 M024-S047-S080 0.00 6.50 M009-S199-S141 0.77 6.39 M018-S136-S1830.39 6.34 M036-S062-S212 1.03 6.30 M012-S211-S137 0.71 6.21M010-S064-S171 0.71 6.19 M042-S141-S120 0.84 6.17 M042-S130-S177 0.846.17 M036-S176-S116 0.58 6.16 M007-S109-S212 0.64 6.14 M010-S195-S1000.84 6.10 M012-S168-S175 0.00 6.07 M018-S102-S117 0.00 6.01M018-S211-S083 0.00 6.00 M018-S192-S058 0.00 6.00 M042-S143-S168 0.005.95 M012-S186-S120 0.00 5.95 M030-S087-S052 0.00 5.95 M008-S186-S0850.00 5.94 M042-S202-S050 0.00 5.93 M001-S171-S214 0.00 5.91M001-S138-S037 0.00 5.91 M012-S143-S176 0.00 5.91 M042-S186-S050 0.005.91 M012-S192-S076 3.09 5.91 M048-S098-S074 0.00 5.90 M042-S186-S2150.00 5.90 M048-S050-S211 0.00 5.90 M010-S074-S169 1.35 5.88M008-S212-S099 3.22 5.88 M001-S074-S197 0.00 5.88

TABLE 11 Library 2.1B Top Constructs. BlockSequence Norm_D7 Enich_D28M042-S186-S044 0.78 8.65 M048-S100-S045 0.98 8.51 M001-S186-S170 2.218.24 M024-S039-S052 6.24 7.53 M001-S214-S058 0.26 7.27 M042-S080-S0742.02 7.04 M030-S170-S115 2.60 6.96 M009-S083-S138 1.04 6.44M042-S154-S075 2.73 6.44 M030-S214-S143 4.49 6.33 M012-S213-S211 1.636.23 M036-S212-S214 0.78 6.17 M030-S036-S106 2.86 6.05 M001-S169-S0831.30 6.04 M007-S105-S064 0.13 5.95 M009-S039-S183 1.56 5.95M009-S074-S050 1.63 5.94 M012-S076-S051 1.43 5.94 M010-S072-S186 1.305.92 M008-S142-S075 0.13 5.91 M001-S175-S168 0.33 5.87 M036-S170-S1750.13 5.87 M018-S194-S116 1.43 5.87 M042-S115-S121 0.33 5.85M018-S043-S086 0.00 5.85 M010-S177-S215 1.63 5.85 M024-S193-S193 0.335.83 M012-S049-S192 0.20 5.80 M048-S130-S170 0.39 5.80 M018-S175-S1711.56 5.80 M030-S051-S080 2.28 5.79 M012-S121-S072 0.13 5.76M009-S183-S161 1.50 5.75 M007-S077-S059 0.33 5.75 M018-S058-S062 1.765.73 M008-S170-S168 0.33 5.73 M018-S050-S043 0.33 5.73 M012-S215-S1261.17 5.69 M024-S083-S047 3.58 5.68 M048-S052-S084 0.52 5.68M042-S052-S130 1.56 5.67 M036-S064-S047 1.95 5.65 M008-S148-S137 0.525.65 M036-S198-S135 0.52 5.64 M018-S043-S051 1.11 5.64 M042-S105-S1040.46 5.62 M030-S074-S082 0.39 5.61 M036-S083-S130 0.52 5.59M001-S137-S157 0.46 5.58 M008-S083-S104 0.72 5.57

TABLE 12 Library 3A Top Constructs. BlockSequence Norm_D7 Enrich_D35BlockSequence Norm_D7 Enrich_D35 E013-T047-S158-S080 0.00 17.08E014-T054-S186-S053 0.00 8.77 E011-T024-S194-S039 0.00 16.99E012-T062-S186-S047 0.92 8.74 E014-T040-S135-S076 0.61 15.47E012-T066-S197-S076 1.34 8.72 E013-T041-S186-S051 2.54 15.08E012-T054-S102-S214 1.18 8.71 E013-T064-S058-S212 3.91 13.86E013-T010-S106-S076 1.03 8.68 E013-T028-S186-S051 12.36 12.80E011-T046-S186-S050 2.26 8.65 E014-T015-S186-S051 0.00 12.78E014-T071-S100-S049 3.43 8.63 E011-T016-S186-S050 2.31 11.97E014-T045-S186-S051 2.26 8.62 E011-T073-S186-S050 0.00 11.96E013-T032-S145-X002 0.00 8.61 E013-T011-S186-S211 1.75 11.90E013-T049-S149-S037 0.00 8.60 E012-T011-S186-S047 3.85 11.85E012-T046-S186-S050 7.48 8.60 E012-T020-S117-S212 0.54 11.33E013-T061-S121-S074 1.50 8.59 E012-T017-S186-S051 0.00 11.31E014-T017-S198-S211 0.94 8.57 E012-T028-S186-S039 0.00 11.22E014-T059-S059-S050 1.08 8.57 E014-T034-S069-S053 0.00 11.19E012-T077-S196-S080 0.00 8.56 E012-T059-S175-S213 3.20 11.05E014-T005-S137-S214 1.09 8.54 E014-T035-S154-S076 2.56 11.04E013-T052-S148-S050 2.08 8.53 E013-T045-S169-S215 4.85 11.02E011-T051-S083-S076 0.00 8.52 E014-T031-S186-S211 2.18 10.86E014-T072-S085-S213 1.13 8.52 E012-T063-S191-S052 0.00 10.75E013-T006-S177-S075 1.63 8.51 E011-T064-S083-S048 2.15 10.67E012-T050-S099-S214 0.00 8.51 E013-T055-S099-S215 0.00 10.65E014-T056-S058-S216 1.10 8.50 E014-T029-S154-S047 0.00 10.64E013-T045-S186-S053 4.11 8.50 E013-T050-S186-S047 0.00 10.62E014-T045-S186-S053 3.15 8.50 E013-T017-S106-S080 1.75 10.54E013-T021-S186-S211 0.00 8.49 E012-T049-S197-S053 1.12 10.44E013-T073-S054-S075 0.00 8.49 E011-T011-S186-S211 5.44 10.40E014-T072-S101-X002 3.23 8.49 E014-T041-S186-S053 3.26 10.35E013-T036-S102-S053 0.00 8.48 E011-T007-S154-S216 0.00 10.33E011-T047-S145-S052 0.00 8.46 E013-T041-S186-S050 11.95 10.28E012-T006-S186-S039 0.00 8.45 E012-T072-S189-S212 0.92 10.18E012-T031-S186-S050 0.95 8.41 E011-T077-S186-S211 5.16 10.13E014-T060-S109-S076 2.22 8.41 E013-T045-S143-S038 0.00 10.11E011-T005-S098-S074 1.12 8.39 E014-T041-S186-X002 3.72 10.09E011-T017-S186-X002 1.05 8.38 E014-T057-S186-S050 3.07 10.09E011-T043-S054-S049 1.04 8.37 E013-T068-S170-S050 1.45 9.91E014-T080-S193-S050 1.01 8.35 E014-T055-S186-S053 0.00 9.90E014-T007-S190-S053 2.44 8.33 E014-T028-S186-S051 2.10 9.86E013-T023-S105-X002 1.09 8.29 E013-T032-S136-S050 0.00 9.83E013-T057-S186-S051 6.29 8.16 E012-T064-S135-S080 1.11 9.80E014-T031-S186-S053 11.27 6.86 E014-T050-S186-S050 1.53 9.80E012-T062-S186-S211 3.99 8.15 E011-T001-S177-S074 6.99 9.74E012-T006-S186-S047 3.85 8.07 E013-T040-S171-S038 2.08 9.73E014-T028-S186-S053 3.49 8.13 E013-T034-S186-S050 0.00 9.72E014-T041-S186-S211 5.72 7.54 E013-T055-S104-S052 0.00 9.50E011-T047-S085-S211 3.66 8.06 E014-T051-S058-S211 3.87 9.48E014-T038-S186-X002 6.13 7.42 E013-T012-S194-X002 0.79 9.42E013-T028-S186-S076 8.76 6.60 E013-T019-S186-S050 3.33 9.42E011-T045-S186-S051 2.25 8.16 E012-T071-S069-S075 0.00 9.42E011-T022-S186-S050 1.93 8.29 E011-T026-S186-S051 1.06 9.34E012-T004-S202-S211 1.91 8.26 E013-T050-S186-S050 0.00 9.29E011-T022-S194-S074 2.04 8.17 E012-T024-S186-S051 0.90 9.27E011-T011-S186-S047 2.19 8.10 E012-T073-S186-S053 0.00 9.12E012-T031-S186-S052 3.62 7.55 E013-T071-S115-S213 1.04 9.09E013-T030-S138-S216 1.95 8.20 E014-T062-S186-S211 2.15 8.99E013-T016-S186-S050 2.98 7.74 E011-T062-S186-S211 0.00 8.92E013-T034-S186-S053 3.51 7.54 E014-T011-S186-S039 0.46 8.86E011-T012-S193-S052 4.66 7.19 E013-T069-S109-S074 0.00 8.84E012-T047-S186-S047 3.51 7.43 E011-T058-S156-X002 0.92 8.83E011-T055-S186-S053 8.63 6.29 E014-T021-S171-S053 0.00 8.83E013-T077-S186-S051 2.48 7.73 E014-T073-S161-S050 0.00 8.81E014-T041-S186-S052 7.19 6.47 E011-T011-S186-S051 1.71 8.79E013-T029-S186-S051 5.53 6.76

TABLE 13 Library 3B Top Constructs. BlockSequence Norm_D7 Enrich_D21BlockSequence Norm_D7 Enrich D21 E014-T011-S186-S039 0.46 9.67E011-T007-S064-S075 0.00 6.58 E013-T057-S115-S214 0.00 9.40E013-T059-S136-S050 1.83 6.58 E013-T028-S186-S051 12.36 9.28E013-T034-S186-S053 3.51 6.56 E013-T019-S142-S212 0.00 9.04E014-T022-S087-S075 0.00 6.55 E011-T020-S104-S080 0.99 8.92E012-T055-S142-X002 0.03 6.55 E011-T011-S186-S039 0.00 8.75E011-T067-S186-S051 1.79 6.53 E013-T031-S169-S214 0.00 8.74E012-T073-S083-S037 4.04 6.48 E013-T062-S158-S051 1.30 8.51E011-T028-S171-S038 1.19 6.47 E011-T041-S186-S047 2.62 8.43E012-T016-S186-S039 0.00 6.45 E012-T024-S142-S211 1.17 8.31E012-T047-S104-S053 1.60 6.44 E013-T017-S143-S216 0.00 8.26E014-T016-S186-S039 2.60 6.37 E013-T058-S183-S051 1.00 8.19E013-T042-S191-S074 1.11 6.37 E011-T030-S176-S050 0.60 8.16E014-T017-S186-S053 0.96 6.33 E012-T062-S186-S037 3.05 8.10E013-T048-S154-S038 0.10 6.33 E014-T041-S085-S037 0.91 8.02E013-T045-S186-S053 4.11 6.32 E011-T080-S115-S211 0.00 7.86E013-T016-S186-S050 2.98 6.27 E013-T041-S186-S039 1.07 7.81E013-T062-S186-S051 1.11 6.23 E014-T028-S186-S053 3.49 7.78E011-T001-S177-S074 6.99 6.23 E014-T019-S057-S074 1.16 7.76E012-T027-S116-S215 0.00 6.22 E012-T044-S130-S074 1.09 7.75E011-T046-S186-S052 0.00 6.21 E013-T081-S085-S211 3.54 7.74E013-T031-S186-S047 3.33 6.10 E014-T028-S186-S051 2.10 7.68E014-T012-S121-S213 1.72 6.09 E012-T027-S083-S080 0.00 7.60E012-T033-S130-X002 0.00 6.09 E013-T052-S126-S213 0.00 7.58E014-T055-S109-S211 0.67 6.06 E012-T039-S176-S213 4.06 7.53E013-T014-S197-S214 0.00 6.02 E014-T072-S101-X002 3.23 7.45E014-T041-S186-S047 0.00 6.02 E012-T011-S186-S047 3.85 7.43E012-T021-S186-S051 1.47 6.01 E014-T041-S186-S053 3.26 7.41E014-T073-S059-S048 0.93 5.99 E013-T002-S135-S038 0.00 7.40E013-T050-S186-S047 0.00 5.98 E011-T015-S170-S047 4.71 7.39E013-T022-S189-S215 1.69 5.97 E014-T072-S198-S076 2.28 7.35E014-T042-S177-X002 6.13 5.95 E014-T028-S186-S039 1.44 7.31E014-T076-S186-S050 8.73 5.95 E013-T027-S186-S051 3.03 7.30E012-T011-S186-S216 0.60 5.93 E014-T011-S186-X002 4.22 7.29E012-T034-S186-S053 3.99 5.92 E014-T015-S186-S051 0.00 7.25E014-T031-S186-S053 11.27 5.70 E012-T010-S063-S039 3.22 7.23E013-T020-S083-X002 7.87 5.69 E011-T011-S186-S053 8.27 7.23E012-T072-S176-S211 6.58 5.74 E012-T067-S072-S075 0.00 7.21E014-T062-S186-X002 11.85 4.83 E012-T043-S186-S051 1.16 7.21E013-T011-S186-S039 5.19 5.75 E012-T030-S059-X002 2.08 7.15E013-T021-S069-S216 4.36 5.82 E011-T011-S186-S051 1.71 7.14E012-T077-S186-S052 5.77 5.42 E012-T059-S175-S213 3.20 7.08E012-T077-S195-S080 4.48 5.64 E011-T038-S116-S039 2.16 7.07E012-T043-S186-S053 8.59 4.72 E012-T028-S186-S039 0.00 7.03E012-T011-S186-S052 4.03 5.63 E012-T024-S186-S053 4.74 6.98E012-T016-S186-S075 7.32 4.87 E012-T033-S157-S053 0.00 6.96E012-T043-S186-X002 3.44 5.73 E013-T071-S072-S213 0.00 6.95E014-T071-S121-X002 3.30 5.77 E012-T050-S099-S214 0.00 6.94E014-T028-S186-X002 5.96 5.03 E011-T062-S186-S053 0.00 6.90E014-T062-S186-S051 4.96 5.24 E013-T044-S143-S050 1.31 6.85E012-T021-S186-S053 17.53 3.59 E014-T008-S186-S053 1.59 6.82E014-T062-S102-S215 5.11 5.13 E014-T065-S142-S050 2.28 6.79E014-T011-S186-S053 10.09 4.26 E013-T055-S195-S076 0.00 6.78E012-T062-S186-S076 3.01 5.70 E013-T028-S186-S047 0.52 6.77E013-T028-S186-S076 8.76 4.42 E014-T077-S130-S038 0.00 6.74E013-T062-S186-S075 2.89 5.73 E014-T016-S186-S051 1.17 6.73E011-T030-S082-X002 8.98 4.37 E013-T039-S186-S214 1.55 6.72E013-T019-S186-S050 3.33 5.51 E012-T012-S103-S213 0.00 6.69E013-T011-S186-S037 3.85 5.33 E013-T032-S142-S074 1.03 6.67E012-T002-S136-S080 6.27 4.74 E013-T057-S186-S051 6.29 6.66E013-T062-S186-S047 2.87 5.61 E012-T078-S183-S212 0.00 6.65E012-T034-S186-S051 6.25 4.70 E013-T046-S135-S037 0.00 6.64E012-T046-S186-S050 7.48 4.37 E013-T008-S101-S080 1.08 6.62E011-T067-S165-S038 6.40 4.56 E012-T044-S105-S214 1.16 6.61E012-T031-S186-X002 4.42 4.95 E014-T001-S186-S053 0.00 6.59E014-T019-S170-X002 3.05 5.35 E013-T028-S186-S050 0.00 6.58

TABLE 14 Library 3.1A Top Constructs. BlockSequence Norm_D7 Enrich_D35E006-T074-S194-S211 0.00 15.37 E010-T073-S186-S211 3.27 14.82E009-T049-S106-S037 0.00 13.34 E009-T073-S190-S215 2.10 13.00E009-T009-S165-S052 0.80 12.98 E009-T066-S168-S053 2.10 12.85E010-T024-S197-S214 0.00 12.59 E009-T072-S186-S039 0.00 12.42E009-T038-S105-S050 0.00 12.38 E006-T057-S105-S039 0.00 12.38E007-T017-S197-S047 1.79 12.25 E010-T045-S138-S074 0.00 12.20E008-T024-S197-S039 0.00 12.16 E006-T077-S202-S053 0.00 12.09E009-T032-S105-S048 1.85 12.05 E008-T056-S197-S050 0.00 12.02E006-T008-S170-S075 1.36 12.01 E010-T017-S084-S052 0.00 11.96E009-T032-S129-S047 0.00 11.69 E010-T053-S194-S211 1.30 11.69E008-T010-S064-S037 0.00 11.68 E009-T060-S175-S053 0.00 11.50E008-T073-S169-X002 0.00 11.39 E009-T071-S186-S211 4.08 11.38E006-T077-S069-S050 4.94 11.35 E007-T065-S106-S053 1.23 11.32E008-T001-S190-S053 0.00 11.30 E007-T032-S138-S052 0.00 11.24E010-T044-S190-S080 2.28 11.20 E009-T044-S129-S053 1.23 11.18E008-T073-S186-S080 0.00 11.17 E007-T020-S082-S211 0.00 11.16E007-T006-S195-S052 0.00 11.11 E008-T027-S190-S050 1.61 11.08E007-T056-S143-S050 0.68 10.99 E010-T042-S177-S049 1.85 10.96E008-T034-S064-S039 0.00 10.95 E007-T040-S054-S052 1.85 10.83E010-T019-S054-S051 1.48 10.80 E007-T058-S086-S052 1.48 10.78E009-T019-S192-S214 0.86 10.69 E010-T066-S192-S037 1.91 10.67E006-T048-S194-S074 0.00 10.57 E006-T078-S102-S075 1.05 10.55E010-T017-S197-S053 1.17 10.55 E010-T041-S169-S074 0.00 10.50E006-T038-S192-S053 1.79 10.47 E007-T032-S129-S212 0.49 10.46E007-T035-S189-S051 4.08 10.45 E006-T006-S116-S214 0.00 10.44

TABLE 15 Library 3.1B Top Constructs. BlockSequence Norm_D7 Enrich_D35E010-T044-S171-S050 0.00 15.69 E007-T061-S169-S211 0.00 15.11E010-T072-S183-S212 3.89 14.56 E008-T013-S192-S037 5.19 14.19E008-T058-S176-S212 6.11 13.64 E006-T045-S130-S039 3.64 13.62E008-T006-S169-X002 5.93 13.43 E008-T041-S141-S053 6.54 13.29E007-T024-S115-S074 8.40 12.97 E009-T021-S138-S037 7.16 12.91E009-T044-S197-S051 7.78 11.62 E009-T019-S192-S214 0.86 11.28E008-T065-S101-X002 1.42 10.48 E008-T082-S102-S048 0.00 10.15E010-T072-S186-S211 2.59 10.14 E007-T006-S141-S050 0.00 10.08E009-T073-S064-S037 0.00 9.96 E010-T072-S197-S051 0.00 9.80E007-T054-S194-S214 0.00 9.72 E010-T044-S137-S052 0.00 9.51E010-T078-S197-S051 0.31 9.40 E007-T003-S120-S075 0.00 9.24E010-T030-S197-S037 0.00 9.22 E007-T017-S058-S211 0.74 9.20E006-T068-S136-S048 0.00 9.15 E006-T034-S192-S049 0.99 9.11E010-T040-S102-S051 0.00 9.11 E009-T041-S129-S049 0.93 9.03E008-T003-S176-S215 0.00 8.92 E006-T055-S105-S214 0.00 8.87E007-T039-S058-S211 0.00 8.79 E010-T020-S104-X002 1.54 8.67E010-T030-S197-S047 0.00 8.65 E007-T038-S099-X002 0.00 8.62E010-T024-S197-S214 0.00 8.59 E008-T041-S058-S216 0.00 8.59E010-T013-S130-S080 1.11 8.56 E008-T079-S168-S038 0.00 8.48E008-T021-S126-S053 0.00 8.39 E010-T045-S102-S213 1.17 8.38E009-T076-S186-S211 0.68 8.36 E009-T005-S196-S049 1.54 8.33E008-T020-S100-S053 0.00 8.25 E009-T075-S102-S039 9.88 8.23E008-T006-S064-S051 0.00 8.20 E007-T046-S154-S075 0.00 8.18E008-T071-S138-S047 0.00 8.17 E006-T008-S170-S075 1.36 8.15E007-T073-S154-S048 0.00 8.13 E008-T001-S190-S053 0.00 8.13

TABLE 16 Library 4B Top Constructs. BlockSequence Norm_D7 Enrich_D21BlockSequence Norm_D7 Enrich_D21 E014-T046-S186- 0.000 11.939E013-T062-S058- 6.678 7.463 E014-T041-S058- 0.000 10.739 E013-T008-S072-2.701 7.451 E012-T067-S142- 0.000 10.684 E012-T030-S130- 0.954 7.443E013-T053-S101- 0.000 10.439 E012-T065-S057- 0.000 7.436 E011-T041-S186-0.000 10.295 E011-T015-S186- 0.000 7.430 E012-T013-S158- 0.000 10.040E014-T028-S186- 0.000 7.417 E013-T029-S058- 4.437 9.654 E011-T071-S098-4.692 7.401 E011-T044-S145- 0.000 9.625 E013-T079-X001- 0.000 7.392E012-T008-S085- 4.537 9.592 E011-T040-S143- 0.000 7.372 E014-T065-S064-1.253 9.559 E012-T006-S186- 0.000 7.362 E011-T047-S189- 0.000 9.513E014-T082-S109- 0.000 7.351 E012-T078-S154- 0.000 9.471 E014-T004-X001-0.000 7.344 E013-T024-S176- 2.385 9.354 E014-T053-S174- 0.000 7.338E012-T022-S186- 0.000 9.344 E012-T023-S135- 0.000 7.320 E012-T016-S186-1.875 9.244 E011-T038-X001- 0.000 7.307 E013-T030-S175- 2.252 9.223E012-T011-S186- 0.000 7.287 E013-T077-S186- 0.000 9.217 E011-T057-S169-0.000 7.284 E012-T048-S157- 0.000 9.153 E012-T015-S186- 0.000 7.275E013-T016-S186- 2.036 9.085 E011-T049-S189- 0.000 7.263 E011-T041-S199-2.779 9.018 E011-T031-S186- 5.596 7.257 E011-T041-S186- 0.000 8.856E013-T029-S104- 0.000 7.234 E012-T022-S186- 0.000 8.856 E013-T041-S186-0.000 7.226 E014-T016-S142- 0.000 8.830 E013-T073-S186- 0.000 7.214E011-T077-S186- 0.000 8.615 E011-T021-S157- 170.218 4.017E013-T011-S186- 5.158 8.540 E014-T027-S176- 174.583 3.959E012-T031-S085- 0.000 8.537 E011-T021-S083- 151.189 3.611E013-T041-S143- 4.731 8.536 E012-T050-S145- 68.520 4.545 E013-T023-S105-0.000 8.515 E011-T067-S146- 49.063 4.896 E012-T011-S186- 2.024 8.510E011-T052-S143- 70.467 4.322 E012-T031-S085- 0.000 8.471 E014-T074-S138-66.035 4.290 E014-T016-S186- 1.137 8.452 E013-T012-S058- 92.824 3.772E013-T069-S115- 0.000 8.437 E012-T015-S069- 86.934 3.776 E011-T008-S177-0.000 8.330 E013-T023-S157- 85.531 3.689 E014-T017-S141- 0.971 8.328E012-T006-S136- 7.798 6.983 E014-T028-S186- 1.897 8.305 E012-T024-S062-69.784 3.940 E013-T016-S186- 0.000 8.292 E012-T025-S085- 108.753 3.034E014-T044-S099- 2.202 8.285 E011-T016-S186- 5.447 7.123 E014-T020-S168-2.235 8.284 E011-T015-S186- 9.296 6.412 E012-T030-S115- 0.000 8.257E012-T004-S195- 44.560 4.178 E014-T002-S169- 0.000 8.239 E011-T032-S102-5.635 6.948 E013-T020-S194- 0.000 8.226 E011-T014-S117- 10.350 6.156E011-T018-S117- 0.000 8.212 E014-T015-S186- 6.839 6.688 E013-T062-S186-7.937 8.203 E013-T048-S177- 60.727 3.700 E014-T062-S186- 0.000 8.155E012-T028-S186- 17.510 5.428 E012-T078-S129- 0.000 8.111 E012-T059-S135-6.206 6.655 E011-T017-S077- 0.000 8.105 E012-T044-S081- 45.663 3.895E011-T006-S186- 0.000 8.095 E014-T011-S186- 4.298 7.022 E013-T050-S186-4.359 8.015 E011-T017-S058- 7.582 6.323 E011-T041-S054- 0.000 7.984E013-T027-S175- 27.643 4.558 E011-T036-S138- 0.000 7.978 E014-T056-S136-4.099 7.044 E014-T080-S197- 0.000 7.899 E014-T065-S196- 4.875 6.823E014-T036-S106- 0.000 7.886 E013-T028-S186- 4.215 6.893 E014-T017-S058-2.263 7.837 E011-T023-S058- 37.543 3.995 E012-T055-S186- 0.932 7.824E011-T028-S186- 5.524 6.499 E011-T015-S186- 0.000 7.806 E014-T072-S069-48.963 3.487 E014-T030-S058- 0.000 7.798 E011-T032-S130- 25.169 4.402E011-T017-S054- 1.764 7.786 E012-T052-S169- 4.099 6.662 E012-T028-S186-1.198 7.779 E012-T076-S121- 38.253 3.711 E013-T062-S186- 0.000 7.749E012-T062-S186- 10.250 5.469 E012-T011-S186- 0.000 7.707 E013-T011-S186-9.972 5.499 E013-T035-S145- 0.000 7.704 E013-T029-S186- 4.215 6.526E012-T066-S130- 0.000 7.701 E013-T011-S186- 4.398 6.474 E012-T017-S192-0.000 7.624 E011-T075-S130- 60.189 2.941 E012-T002-S084- 0.998 7.614E014-T044-S193- 2.302 7.150 E013-T050-S138- 0.000 7.603 E013-T045-S186-5.175 6.203 E013-T043-S186- 0.000 7.595 E011-T065-S143- 28.447 3.947E011-T034-S064- 0.000 7.589 E013-T077-S099- 2.191 7.134 E012-T017-S186-0.072 7.567 E014-T057-S186- 3.627 6.594 E011-T030-S186- 0.000 7.564E013-T011-S186- 6.678 5.862 E012-T011-S170- 0.000 7.560 E011-T073-S069-4.104 6.445 E012-T039-S177- 0.000 7.540 E012-T046-S186- 7.621 5.686E014-T041-S186- 2.158 7.534 E014-T062-S186- 5.940 5.989 E014-T012-S183-0.000 7.513 E014-T028-S186- 2.074 7.146 E013-T055-S186- 0.000 7.509E014-T055-S186- 7.565 5.614 E012-T045-S170- 0.000 7.487 E014-T011-S186-8.885 5.336 E012-T024-S186- 0.000 7.469 E013-T055-S186- 2.202 6.852E013-T010-S183- 0.000 7.463 E011-T019-S072- 26.351 3.742

TABLE 17 Library 4.1B Top Constructs. BlockSequence Norm_D7 Enrich_D42E009-T061-S170-S050 0.56 9.29 E008-T001-S190-S047 0.00 8.97E008-T020-S083-S080 0.77 8.64 E009-T074-S085-X002 1.39 8.43E010-T034-S058-S212 0.70 8.13 E006-T010-S064-S051 0.42 7.90E009-T055-S064-S053 1.05 7.73 E006-T039-S193-S075 0.70 7.38E008-T071-S109-S212 1.12 7.37 E010-T019-S168-X002 1.81 7.31E009-T072-S170-S211 0.77 6.56 E006-T049-S145-S052 0.28 6.52E009-T010-S064-S051 2.02 6.49 E007-T010-S064-S047 1.26 6.41E007-T056-S180-S213 1.32 6.33 E007-T008-S154-S074 0.28 6.31E010-T027-S199-S075 0.07 6.31 E010-T017-S136-S050 0.14 6.26E008-T043-S109-S047 0.35 6.20 E008-T038-S102-S051 0.14 6.13E008-T017-S058-X002 0.70 6.13 E007-T065-S102-S049 1.32 6.06E006-T030-S084-S074 1.53 6.03 E010-T036-S192-S050 0.70 5.88E009-T017-S168-S216 0.49 5.87 E008-T079-S142-S211 0.00 5.84E008-T082-S126-S053 0.21 5.82 E010-T022-S143-S213 0.70 5.78E009-T006-S194-S038 0.28 5.76 E006-T077-S199-S075 0.21 5.76E006-T002-S198-X002 0.70 5.74 E010-T002-S165-S052 0.70 5.72E010-T079-S194-S053 0.14 5.68 E006-T019-S199-S076 0.07 5.68E008-T053-S102-S037 0.35 5.65 E006-T069-S193-S038 0.07 5.65E008-T072-S171-S212 0.91 5.63 E007-T062-S137-S214 0.28 5.60E007-T060-S176-S047 0.21 5.60 E009-T034-S064-S051 1.19 5.58E008-T078-S103-S211 0.07 5.57 E008-T010-S062-S051 0.56 5.56E010-T057-S154-S213 0.28 5.55 E010-T051-S138-S075 0.28 5.54E010-T031-S102-S039 0.07 5.48 E009-T071-S180-S074 0.14 5.48E009-T027-S175-S213 0.00 5.46 E007-T052-S193-S214 1.46 5.45E009-T030-S087-S039 0.56 5.45 E007-T006-S059-S047 0.35 5.44

TABLE 18 Lib3 P4_STOP. BlockSequence N_D7 N_D21 3B E_D21 3B N_D21 3AE_D21 3A N_D35 3A E_D35 3A E014-T041-S186- 3.72 146.55 4.97 4927.5410.03 5163.05 10.09 E013-T012-S194- 0.79 16.46 3.29 268.84 7.24 1221.60 9.42 E011-T058-S156- 0.92 41.10 4.45 275.82 7.17 874.92  8.83E013-T032-S145- 0.00 0.00 0.00 613.68 9.26 390.18  8.61 E014-T072-S101-3.23 740.94 7.45 14542.93 11.75 1520.08  8.49 E011-T017-S186- 1.05 0.00−1.04 1960.69 9.90 680.44  8.38 E013-T023-S105- 1.09 0.00 −1.07 151.636.19 655.49  8.29 E011-T039-S176- 1.12 20.25 3.32 742.40 8.45 504.52 7.89 E013-T055-S194- 0.00 16.40 4.12 1736.80 10.76 191.90  7.59E014-T038-S186- 6.13 0.00 −2.83 1147.62 7.33 1218.74  7.42E014-T019-S170- 3.05 164.40 5.35 950.31 7.87 665.15  7.36E014-T014-S110- 0.00 0.00 0.00 269.39 8.08 133.71  7.07 E013-T045-S135-2.82 0.00 −1.94 39.44 3.40 460.88  6.92 E013-T035-S176- 0.00 34.31 5.14216.60 7.77 105.91  6.74 E014-T067-S106- 2.21 118.19 5.21 541.72 7.40322.27  6.65 E012-T020-S186- 3.03 26.98 2.80 5712.32 10.47 326.42  6.35E014-T074-S199- 1.17 0.00 −1.12 90.02 5.39 146.76  6.09 E013-T066-S194-0.00 9.85 3.44 9.12 3.34 52.14  5.73 E012-T030-S059- 2.08 437.19 7.151336.66 8.76 142.82  5.54 E011-T057-S102- 1.18 9.13 2.22 0.00 −1.1299.52  5.53 E013-T075-S116- 0.95 0.00 −0.96 551.34 8.14 86.06  5.48E011-T063-S158- 1.45 0.00 −1.29 218.81 6.49 106.86  5.46 E013-T057-S120-0.00 17.18 4.18 33.01 5.09 42.82  5.45 E012-T033-S130- 0.00 67.18 6.09142.99 7.17 41.19  5.40 E014-T067-S194- 0.86 79.68 5.44 342.51 7.5367.16  5.20 E014-T032-S197- 0.45 10.58 2.99 166.33 6.85 48.53  5.09E012-T017-S059- 2.72 15.38 2.14 66.93 4.19 125.01  5.08 E014-T022-S130-0.00 19.29 4.34 132.58 7.06 29.71  4.94 E012-T003-S130- 0.00 16.46 4.13316.91 8.31 27.19  4.82 E011-T030-S082- 8.98 204.84 4.37 1073.77 6.75277.27  4.80 E012-T043-S116- 2.15 0.00 −1.66 9.74 1.77 79.53  4.68E014-T028-S186- 5.96 226.89 5.03 2380.18 8.42 153.76  4.47E012-T064-S168- 2.07 6.61 1.31 263.63 6.43 58.60  4.28 E013-T028-S117-0.00 0.00 0.00 147.22 7.21 17.67  4.22 E014-T014-S106- 1.39 9.07 2.070.00 −1.26 42.21  4.17 E012-T064-S072- 0.96 0.00 −0.97 36.87 4.27 33.92 4.15 E013-T071-S117- 0.00 0.00 0.00 68.83 6.13 16.18  4.10E013-T020-S083- 7.87 458.11 5.69 1612.42 7.51 148.53  4.08E013-T009-S195- 1.08 8.65 2.22 0.00 −1.05 33.58  4.06 E012-T074-S143-0.73 8.35 2.44 89.35 5.71 26.65  4.00 E013-T007-S084- 0.00 0.00 0.0013.23 3.83 14.89  3.99 E013-T031-S165- 2.94 10.58 1.55 45.56 3.56 60.02 3.95 E013-T016-S186- 4.90 113.99 4.29 584.77 6.63 87.76  3.91E011-T021-S104- 0.00 9.55 3.40 97.43 6.62 14.00  3.91 E012-T001-X001-5.69 8.65 0.53 645.09 6.59 92.65  3.81 E013-T041-S186- 15.80 41.16 1.33286.42 4.10 234.04  3.81 E014-T011-S186- 4.22 814.84 7.29 2615.15 8.9771.51  3.80 E013-T007-S121- 2.12 10.34 1.86 7.29 1.41 39.90  3.71E012-T037-X001- 1.28 0.00 −1.19 15.49 2.85 28.82  3.71 E014-T071-S121-3.30 233.32 5.77 469.83 6.77 55.13  3.71 E013-T057-S176- 8.49 10.03 0.22131.66 3.81 114.27  3.60 E014-T011-S170- 2.73 0.00 −1.90 484.03 7.0243.91  3.59 E014-T044-S190- 1.73 9.97 2.00 149.91 5.79 31.00  3.55E012-T055-S168- 1.19 0.00 −1.13 154.51 6.15 24.54  3.54 E014-T068-S121-0.00 19.53 4.36 115.93 6.87 10.40  3.51 E012-T043-S186- 3.44 234.64 5.73434.80 6.62 49.42  3.50 E012-T060-S195- 4.58 26.26 2.29 150.89 4.7760.91  3.47 E014-T017-S099- 2.26 12.50 2.05 193.70 5.90 33.92  3.42E014-T047-S189- 0.55 4.09 1.71 13.47 3.22 15.50  3.41 E014-T062-S186-11.85 364.97 4.83 1056.62 6.36 135.27  3.41 E014-T069-S103- 1.86 0.00−1.52 136.56 5.59 28.89  3.39 E012-T041-S190- 3.43 10.52 1.38 105.394.58 43.84  3.34 E014-T046-S186- 3.96 0.00 −2.31 471.60 6.57 46.29  3.25E014-T079-S085- 0.97 9.97 2.48 107.90 5.79 17.40  3.22 E014-T032-S142-2.26 6.13 1.13 164.98 5.67 29.03  3.20 E013-T021-S157- 5.66 0.00 −2.74130.01 4.30 60.16  3.20 E012-T031-S186- 4.42 166.56 4.95 882.03 7.3547.86  3.17 E014-T035-S117- 0.00 0.00 0.00 51.75 5.72 7.34  3.06E011-T043-S183- 1.84 0.00 −1.50 0.00 −1.50 19.85  2.88 E011-T045-S147-0.19 5.17 2.37 12.43 3.50 7.27  2.80 E013-T003-S104- 3.48 101.07 4.51391.99 6.46 29.71  2.78 E014-T006-S054- 1.13 0.00 −1.09 0.00 −1.09 11.76 2.58 E013-T073-S126- 0.00 7.51 3.09 7.78 3.13 4.96  2.58E012-T057-S186- 2.73 29.74 3.04 210.23 5.82 20.66  2.54 E014-T015-S165-2.19 0.00 −1.67 0.00 −1.67 17.33  2.52 E014-T010-S147- 0.57 0.00 −0.6536.25 4.57 7.89  2.50 E011-T012-S176- 1.95 7.75 1.57 0.00 −1.56 15.43 2.48 E014-T078-S116- 0.93 4.03 1.38 92.72 5.60 9.58  2.46E013-T051-S120- 5.29 11.30 0.97 89.23 3.84 33.04  2.44 E014-T072-S129-2.37 20.07 2.65 88.06 4.73 15.16  2.26 E013-T001-S101- 0.93 0.00 −0.950.00 −0.95 7.48  2.13 E012-T019-S062- 1.19 0.00 −1.13 89.35 5.37 8.56 2.13 E012-T015-S186- 2.88 0.00 −1.96 0.00 −1.96 14.61  2.01E011-T055-S186- 3.65 41.34 3.19 27.99 2.64 17.54  2.00 E013-T021-S165-2.06 0.00 −1.61 410.18 7.07 10.20  1.87 E012-T056-S143- 2.34 0.00 −1.7478.02 4.56 10.94  1.84 E014-T016-S186- 8.46 36.29 1.98 483.36 5.68 32.02 1.80 E012-T069-S165- 2.12 0.00 −1.64 309.01 6.64 9.65  1.77E013-T034-S186- 4.33 0.00 −2.41 64.55 3.62 15.84  1.66 E012-T057-S183-5.58 25.90 2.03 18.25 1.55 17.06  1.46 E014-T057-S126- 1.87 0.00 −1.5230.07 3.43 6.12  1.31 E013-T010-S054- 6.44 40.50 2.48 122.11 4.05 16.11 1.20 E012-T011-S186- 10.02 87.67 3.01 424.02 5.27 22.09  1.07E014-T050-S186- 7.72 31.31 1.89 59.95 2.81 15.63  0.93 E013-T027-S194-5.15 42.12 2.81 167.98 4.78 9.31  0.75 E014-T028-S191- 1.72 46.09 4.1268.40 4.67 2.99  0.55 E011-T062-S083- 0.00 0.00 0.00 45.26 5.53 0.07 0.09 E011-T001-S081- 0.00 8.17 3.20 0.00 0.00 0.00  0.00E011-T001-S171- 0.00 0.00 0.00 6.74 2.95 0.00  0.00 E011-T001-S183- 0.000.00 0.00 6.92 2.99 0.00  0.00 E011-T004-S082- 0.00 9.37 3.37 0.00 0.000.00  0.00 E011-T004-S126- 0.00 0.00 0.00 5.33 2.66 0.00  0.00E011-T004-S169- 0.00 0.00 0.00 8.82 3.30 0.00  0.00 E011-T005-S086- 0.008.41 3.23 0.00 0.00 0.00  0.00 E011-T005-S121- 0.00 6.91 2.98 0.00 0.000.00  0.00 E011-T005-S129- 0.00 3.55 2.18 0.00 0.00 0.00  0.00E011-T006-S168- 0.00 19.77 4.38 42.38 5.44 0.00  0.00 E011-T006-S189-0.00 9.61 3.41 0.00 0.00 0.00  0.00 E011-T008-S077- 0.00 9.07 3.33 0.000.00 0.00  0.00 E011-T008-S104- 0.00 0.00 0.00 5.45 2.69 0.00  0.00E011-T008-S117- 0.00 10.03 3.46 0.00 0.00 0.00  0.00 E011-T009-S077-0.00 8.89 3.31 0.00 0.00 0.00  0.00 E011-T009-S145- 0.00 7.45 3.08 0.000.00 0.00  0.00 E011-T011-S058- 0.00 9.55 3.40 0.00 0.00 0.00  0.00E011-T011-S186- 0.00 21.69 4.50 142.75 7.17 0.00  0.00 E011-T012-S085-0.00 8.59 3.26 0.00 0.00 0.00  0.00 E011-T012-S149- 0.00 0.00 0.00 28.724.89 0.00  0.00 E011-T012-S186- 0.00 9.55 3.40 0.00 0.00 0.00  0.00E011-T014-S104- 0.00 0.00 0.00 8.21 3.20 0.00  0.00 E011-T014-S180- 0.0010.46 3.52 0.00 0.00 0.00  0.00 E011-T016-S157- 0.00 9.13 3.34 0.00 0.000.00  0.00 E011-T016-S175- 0.00 5.89 2.78 0.00 0.00 0.00  0.00E011-T018-S129- 0.00 10.88 3.57 0.00 0.00 0.00  0.00 E011-T018-S186-0.00 8.29 3.22 0.00 0.00 0.00  0.00 E011-T019-S137- 0.00 9.61 3.41 0.000.00 0.00  0.00 E011-T019-S143- 0.00 9.79 3.43 0.00 0.00 0.00  0.00E011-T019-S168- 0.00 4.45 2.45 0.00 0.00 0.00  0.00 E011-T019-S175- 0.009.43 3.38 0.00 0.00 0.00  0.00 E011-T020-S156- 0.00 9.73 3.42 0.00 0.000.00  0.00 E011-T021-S103- 0.00 4.27 2.40 0.00 0.00 0.00  0.00E011-T022-S069- 0.00 9.31 3.37 0.00 0.00 0.00  0.00 E011-T023-S084- 0.005.11 2.61 0.00 0.00 0.00  0.00 E011-T023-S143- 0.00 9.91 3.45 0.00 0.000.00  0.00 E011-T027-S058- 0.00 4.81 2.54 0.00 0.00 0.00  0.00E011-T027-S186- 0.00 0.00 0.00 19.11 4.33 0.00  0.00 E011-T028-S186-0.00 3.06 2.02 143.48 7.17 0.00  0.00 E011-T029-S101- 0.00 7.93 3.160.00 0.00 0.00  0.00 E011-T029-S136- 0.00 10.09 3.47 0.00 0.00 0.00 0.00 E011-T030-S109- 0.00 8.83 3.30 0.00 0.00 0.00  0.00E011-T030-S176- 0.00 9.01 3.32 0.00 0.00 0.00  0.00 E011-T031-S191- 0.009.55 3.40 7.41 3.07 0.00  0.00 E011-T032-S175- 0.00 8.71 3.28 0.00 0.000.00  0.00 E011-T033-S059- 0.00 7.09 3.02 0.00 0.00 0.00  0.00E011-T033-S117- 0.00 6.97 2.99 0.00 0.00 0.00  0.00 E011-T033-X001- 0.008.29 3.22 0.00 0.00 0.00  0.00 E011-T034-S137- 0.00 8.77 3.29 0.00 0.000.00  0.00 E011-T034-S170- 0.00 0.00 0.00 7.10 3.02 0.00  0.00E011-T035-S176- 0.00 9.49 3.39 0.00 0.00 0.00  0.00 E011-T037-S106- 0.000.00 0.00 6.98 3.00 0.00  0.00 E011-T038-S077- 0.00 9.01 3.32 0.00 0.000.00  0.00 E011-T038-S103- 0.00 8.41 3.23 0.00 0.00 0.00  0.00E011-T039-S054- 0.00 8.83 3.30 0.00 0.00 0.00  0.00 E011-T039-S137- 0.0010.52 3.53 0.00 0.00 0.00  0.00 E011-T040-S190- 0.00 9.37 3.37 0.00 0.000.00  0.00 E011-T041-S117- 0.00 28.90 4.90 49.97 5.67 0.00  0.00E011-T041-S169- 0.00 7.33 3.06 0.00 0.00 0.00  0.00 E011-T041-S171- 0.008.95 3.32 0.00 0.00 0.00  0.00 E011-T041-S189- 0.00 9.43 3.38 0.00 0.000.00  0.00 E011-T041-X001- 0.00 9.07 3.33 3.49 2.17 0.00  0.00E011-T042-S105- 0.00 8.89 3.31 0.00 0.00 0.00  0.00 E011-T042-S117- 0.009.49 3.39 0.00 0.00 0.00  0.00 E011-T042-S126- 0.00 9.37 3.37 0.00 0.000.00  0.00 E011-T044-S106- 0.00 9.85 3.44 0.00 0.00 0.00  0.00E011-T044-S165- 0.00 0.00 0.00 6.61 2.93 0.00  0.00 E011-T045-S120- 0.009.25 3.36 0.00 0.00 0.00  0.00 E011-T045-S143- 0.00 9.25 3.36 6.61 2.930.00  0.00 E011-T046-S102- 0.00 0.00 0.00 21.37 4.48 0.00  0.00E011-T047-S146- 0.00 4.51 2.46 0.00 0.00 0.00  0.00 E011-T047-S192- 0.000.00 0.00 29.33 4.92 0.00  0.00 E011-T048-S058- 0.00 8.11 3.19 0.00 0.000.00  0.00 E011-T048-S104- 0.00 7.87 3.15 0.00 0.00 0.00  0.00E011-T050-S156- 0.00 17.55 4.21 0.00 0.00 0.00  0.00 E011-T050-S161-0.00 8.05 3.18 0.00 0.00 0.00  0.00 E011-T050-S186- 0.00 0.00 0.00 20.454.42 0.00  0.00 E011-T050-S197- 0.00 8.47 3.24 0.00 0.00 0.00  0.00E011-T051-S130- 0.00 9.19 3.35 0.00 0.00 0.00  0.00 E011-T052-S143- 0.0010.58 3.53 0.00 0.00 0.00  0.00 E011-T053-S102- 0.00 10.40 3.51 0.000.00 0.00  0.00 E011-T054-S058- 0.00 9.73 3.42 0.00 0.00 0.00  0.00E011-T054-S129- 0.00 0.00 0.00 4.10 2.35 0.00  0.00 E011-T055-S168- 0.009.67 3.42 0.00 0.00 0.00  0.00 E011-T058-S145- 0.00 0.00 0.00 28.97 4.910.00  0.00 E011-T061-S158- 0.00 0.00 0.00 16.72 4.15 0.00  0.00E011-T061-S177- 0.00 6.85 2.97 0.00 0.00 0.00  0.00 E011-T061-S193- 0.008.53 3.25 0.00 0.00 0.00  0.00 E011-T063-S171- 0.00 9.85 3.44 0.00 0.000.00  0.00 E011-T065-S064- 0.00 11.72 3.67 0.00 0.00 0.00  0.00E011-T065-S100- 0.00 8.29 3.22 0.00 0.00 0.00  0.00 E011-T065-S102- 0.009.19 3.35 0.00 0.00 0.00  0.00 E011-T066-X001- 0.00 9.07 3.33 0.00 0.000.00  0.00 E011-T067-S148- 0.00 0.00 0.00 50.15 5.68 0.00  0.00E011-T068-S102- 0.00 9.85 3.44 0.00 0.00 0.00  0.00 E011-T068-S176- 0.009.01 3.32 0.00 0.00 0.00  0.00 E011-T069-S103- 0.00 9.67 3.42 0.00 0.000.00  0.00 E011-T069-S171- 0.00 0.00 0.00 7.72 3.12 0.00  0.00E011-T070-S069- 0.00 8.05 3.18 0.00 0.00 0.00  0.00 E011-T072-S191- 0.009.85 3.44 0.00 0.00 0.00  0.00 E011-T073-S120- 0.00 5.71 2.75 0.00 0.000.00  0.00 E011-T074-S062- 0.00 8.59 3.26 0.00 0.00 0.00  0.00E011-T075-S195- 0.00 7.27 3.05 4.04 2.33 0.00  0.00 E011-T079-S135- 0.009.19 3.35 0.00 0.00 0.00  0.00 E011-T080-S069- 0.00 8.89 3.31 0.00 0.000.00  0.00 E011-T080-S135- 0.00 22.23 4.54 0.00 0.00 0.00  0.00E011-T081-S102- 0.00 7.03 3.01 0.00 0.00 0.00  0.00 E012-T001-S155- 0.000.00 0.00 6.00 2.81 0.00  0.00 E012-T001-S157- 0.00 8.71 3.28 0.00 0.000.00  0.00 E012-T002-S069- 0.00 0.00 0.00 3.18 2.07 0.00  0.00E012-T002-S101- 0 7.390728973 3.068796155 0 0 0  0 E012-T002-S137- 0 0 03.551864665 2.186457664 0  0 E012-T003-X001- 0 0 0 4.225494172.385567478 0  0 E012-T004-S154- 0 3.7254081 2.24043893 0 0 0  0E012-T004-S196- 0 10.09465421 3.471792798 0 0 0  0 E012-T006-S054- 0 0 015.61595672 4.054497458 0  0 E012-T007-S137- 0 0 0 198.78194317.642282384 0  0 E012-T007-S183- 0 0 0 5.572753181 2.71649781 0  0E012-T008-S059- 0 8.832822432 3.29760559 0 0 0  0 E012-T008-S165- 0 0 06.552577916 2.916969163 0  0 E012-T009-S126- 0 4.686803739 2.507618015 00 0  0 E012-T009-S199- 0 10.09465421 3.471792798 0 0 0  0E012-T010-S105- 0 7.330641746 3.058427637 0 0 0  0 E012-T010-S180- 0.008.65 3.27 0.00 0.00 0.00  0.00 E012-T011-S059- 0.00 0.00 0.00 77.53 6.300.00  0.00 E012-T011-S062- 0.00 9.07 3.33 0.00 0.00 0.00  0.00E012-T011-S100- 0 8.892909659 3.306394903 0 0 0  0 E012-T011-S157- 07.030205609 3.005436928 0 0 0  0 E012-T011-S158- 0 3.965757012.312013668 0 0 0  0 E012-T012-S116- 0 10.03456698 3.463958111 0 0 0  0E012-T012-S137- 0 4.746890967 2.522781678 0 0 0  0 E012-T012-S146- 09.613956388 3.407890621 0 0 0  0 E012-T013-S115- 0 0 0 6.2463826862.857260997 0  0 E012-T014-S082- 0 8.352124612 3.225294153 0 0 0  0E012-T014-S194- 0 0 0 6.307621732 2.869401957 0  0 E012-T016-S146- 07.751252338 3.129489487 0 0 0  0 E012-T016-S155- 0 5.2876760142.652526881 0 0 0  0 E012-T017-S198- 0 8.292037385 3.215994959 0 0 0  0E012-T019-S145- 0 8.111775703 3.187732234 0 0 0  0 E012-T019-S165- 0 0 08.022315018 3.173497659 0  0 E012-T019-S176- 0 8.352124612 3.2252941537.716119789 3.123686024 0  0 E012-T020-S168- 0 9.613956388 3.4078906215.021601767 2.5901473 0  0 E012-T020-S195- 0 7.751252338 3.129489487 0 00  0 E012-T021-S105- 0 10.57535203 3.532984164 0 0 0  0 E012-T021-S116-0 9.133258569 3.341026273 0 0 0  0 E012-T022-S116- 0 8.7727352043.288762402 0 0 0  0 E012-T022-S120- 0 11.05604985 3.591685382 0 0 0  0E012-T022-S186- 0 0 0 28.41491732 4.878476076 0  0 E012-T023-S109- 0 0 06.552577916 2.916969163 0  0 E012-T023-S189- 0 10.57535203 3.532984164 00 0  0 E012-T024-S054- 0 8.352124612 3.225294153 0 0 0  0E012-T024-S186- 0 0 0 24.9242917 4.696232667 0  0 E012-T025-S059- 08.352124612 3.225294153 0 0 0  0 E012-T026-S069- 0 0 0 6.491338872.905223584 0  0 E012-T026-S130- 0 9.253433024 3.358035124 0 0 0  0E012-T026-S155- 0 0 0 6.736295054 2.951642819 0  0 E012-T026-S169- 04.987239876 2.581891072 0 0 0  0 E012-T029-S169- 0 10.395090343.510340458 0 0 0  0 E012-T030-S099- 0 0 0 12.7989606 3.786487696 0  0E012-T030-S183- 0 4.20610592 2.380204664 0 0 0  0 E012-T030-S193- 05.167501559 2.624686175 0 0 0  0 E012-T031-S098- 0 0 0 7.1037293293.018585988 0  0 E012-T031-S176- 0 9.373607479 3.374845781 0 0 0  0E012-T032-S109- 0 7.93151402 3.158904754 0 0 0  0 E012-T032-S130- 07.871426793 3.149166152 0 0 0  0 E012-T032-S137- 0 4.1460186922.363456698 0 0 0  0 E012-T032-S174- 0 10.27491589 3.495044766 0 0 0  0E012-T032-S176- 0 9.974479753 3.456080645 0 0 0  0 E012-T034-S098- 08.892909659 3.306394903 0 0 0  0 E012-T034-S142- 0 11.17622433.605994935 0 0 0  0 E012-T034-S143- 0 10.39509034 3.510340458 0 0 0  0E012-T036-S168- 0 8.472299067 3.243714632 18.55543092 4.289497422 0  0E012-T038-S175- 0 9.013084114 3.3238145 0 0 0  0 E012-T041-S121- 09.313520251 3.366464938 0 0 0  0 E012-T042-S174- 0 3.8455825552.276670123 0 0 0  0 E012-T042-S198- 0 7.93151402 3.158904754 0 0 0  0E012-T043-S142- 0 10.03456698 3.463958111 0 0 0  0 E012-T043-S192- 03.845582555 2.276670123 0 0 0  0 E012-T043-S194- 0 8.4722990673.243714632 0 0 0  0 E012-T044-S082- 0 10.09465421 3.471792798 0 0 0  0E012-T045-S190- 0 9.734130843 3.424133474 0 0 0  0 E012-T046-S083- 05.528024923 2.706646565 0 0 0  0 E012-T046-S126- 0 0 0 7.1649683753.029447298 0  0 E012-T047-S155- 0 5.347763241 2.666248319 0 0 0  0E012-T048-S165- 0 8.472299067 3.243714632 0 0 0  0 E012-T049-S084- 0 0 014.02374152 3.909172242 0  0 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8.772735204 2.403008377 0 — 0 —E012-T030-S130- 0.847730086 8.892909659 2.420640877 0 — 0 —E014-T002-S106- 0.847730086 0 — 7.593641697 2.217515601 0 —E012-T033-S135- 0.85205524 18.6270405 3.405643616 0 — 0 —E013-T030-S069- 0.85205524 10.21482866 2.598208545 0 — 0 —E011-T077-S130- 0.856380393 11.1762243 2.71350257 0 — 0 —E014-T012-S161- 0.856380393 18.74721496 3.411084927 351.69584087.569788388 0 — E012-T079-S136- 0.860705547 16.40381309 3.225481753 0−0.89584977 0 −0.89584977 E014-T022-S171- 0.860705547 9.5538691612.503850329 0 −0.89584977 0 −0.89584977 E014-T049-S138- 0.8607055477.871426793 2.253316383 0 −0.89584977 0 −0.89584977 E013-T055-S141-0.873681007 9.974479753 2.550205289 0 — 0 — E013-T048-S136- 0.87800616110.03456698 2.554756316 0 — 0 — E014-T049-S102- 0.878006161 28.962043623.995862328 0 — 0 — E011-T032-S174- 0.882331314 0 — 14.819849123.071143352 0 — E012-T074-S196- 0.882331314 8.712647977 2.367344091 0 —0 — E011-T072-S136- 0.886656468 8.832822432 2.381773837 0 — 0 —E013-T047-S116- 0.886656468 8.292037385 2.300163205 0 — 0 —E013-T044-S106- 0.890981621 10.27491589 2.575909425 0 — 0 —E013-T055-S115- 0.890981621 9.373607479 2.45571044 0 — 0 —E014-T042-S085- 0.890981621 7.270554519 2.12884872 0 — 0 —E012-T047-S116- 0.895306775 11.41657321 2.711763779 0 — 0 —E012-T063-S121- 0.895306775 9.133258569 2.418594891 0 — 0 —E013-T014-S084- 0.895306775 7.811339565 2.216929983 0 — 0 —E014-T069-S106- 0.895306775 10.5152648 2.6030443 0 — 0 — E014-T075-S141-0.895306775 8.41221184 2.312102409 0 — 0 — E011-T030-S120- 0.899631928 0−0.92571991 18.8003871 3.381736821 0 −0.92571991 E013-T072-S082-0.899631928 8.592473522 2.336182968 0 −0.92571991 0 −0.92571991E014-T041-S087- 0.899631928 17.78581932 3.305852224 0 −0.92571991 0−0.92571991 E013-T014-S064- 0.903957082 8.772735204 2.359761444 0 — 0 —E011-T011-X001- 0.908282235 10.15474144 2.547310606 0 — 0 —E014-T049-S059- 0.908282235 8.892909659 2.374120341 0 — 0 —E011-T045-S142- 0.912607389 7.330641746 2.122886883 0 — 0 —E012-T016-S137- 0.912607389 9.79421807 2.49664608 0 — 0 —E013-T031-S069- 0.912607389 9.734130843 2.48859272 0 — 0 —E013-T055-S186- 0.912607389 7.150380064 2.091326582 14.023741522.973631488 0 — E012-T013-S098- 0.916932542 8.292037385 2.27719539 0 — 0— E012-T025-S104- 0.916932542 0 — 8.573466432 2.320241832 0 —E014-T012-S129- 0.916932542 9.734130843 2.485333905 0 — 0 —E012-T035-S138- 0.921257696 8.952996887 2.373079954 0 — 0 —E013-T069-S106- 0.921257696 11.8371838 2.740205798 0 — 0 —E014-T038-S103- 0.921257696 7.991601248 2.226527019 0 — 0 —E012-T021-S137- 0.925582849 9.133258569 2.395731076 0 — 0 —E012-T030-S141- 0.929908003 9.79421807 2.483654757 0 — 0 —E013-T017-S194- 0.93 9.07 2.38 0.00 −0.95 0.00 −0.95 E013-T051-S176-0.93 8.83 2.35 0.00 −0.95 0.00 −0.95 E013-T065-S121- 0.93 14.60 3.020.00 −0.95 0.00 −0.95 E014-T055-S100- 0.94 9.79 2.48 0.00 −0.95 0.00−0.95 E012-T077-S103- 0.94 8.41 2.28 0.00 −0.96 0.00 −0.96E013-T051-S109- 0.94 10.64 2.58 0.00 −0.96 0.00 −0.96 E012-T034-S193-0.95 9.67 2.45 0.00 −0.96 0.00 −0.96 E012-T048-S087- 0.951533779.914392525 2.483551502 0 — 0 — E012-T053-S109- 0.95153377 8.171862932.232606367 0 — 0 — E013-T074-S130- 0.95153377 7.691165111 2.154941165 0— 0 — E012-T031-S169- 0.955858924 8.892909659 2.33859259 0 — 0 —E012-T064-S099- 0.955858924 13.33936449 2.874106869 0 — 0 —E012-T012-S189- 0.960184077 10.75561371 2.584288811 0 — 0 —E014-T041-S063- 0.960184077 8.352124612 2.254305012 0 — 0 —E014-T059-S202- 0.960184077 8.892909659 2.335405761 0 — 0 —E011-T042-S109- 0.964509231 8.712647977 2.305695727 0 — 0 —E012-T030-S062- 0.964509231 10.69552648 2.573715955 0 — 0 —E014-T050-S136- 0.964509231 12.55823053 2.786928055 7.2262074212.066058503 0 — E012-T073-S186- 0.968834384 8.892909659 2.329053144 0 —0 — E013-T082-S116- 0.968834384 18.56695327 3.313005471 0 — 0 —E014-T055-S103- 0.968834384 18.44677882 3.304117542 0 — 0 —E014-T058-S135- 0.968834384 8.41221184 2.257192033 0 — 0 —E012-T007-S142- 0.973159538 10.27491589 2.514537158 0 — 0 —E013-T052-S126- 0.973159538 20.30948287 3.432916069 0 — 0 —E012-T032-S183- 0.981809845 10.75561371 2.568459411 0 — 0 —E012-T054-S098- 0.981809845 8.652560749 2.284093187 0 — 0 —E013-T046-S077- 0.981809845 8.772735204 2.30194386 0 — 0 —E013-T046-S137- 0.981809845 7.811339565 2.152542823 0 — 0 —E014-T009-S109- 0.981809845 8.952996887 2.32831245 0 — 0 —E014-T018-S169- 0.981809845 10.81570094 2.575814769 0 — 0 —E014-T032-S198- 0.981809845 30.16378817 3.974980166 0 — 0 —E011-T010-S186- 0.986134998 0 — 8.083554064 2.193293195 0 —E011-T078-S109- 0.986134998 9.493781933 2.401499124 0 — 0 —E013-T059-X001- 0.986134998 7.871426793 2.159202465 0 — 0 —E014-T003-S154- 0.986134998 10.69552648 2.557921215 0 — 0 —E014-T044-S085- 0.986134998 9.433694706 2.393214534 0 — 0 —E012-T018-S202- 0.990460152 1.742529595 0.4624052 7.3486855132.068447076 0 — E012-T080-S145- 0.990460152 9.914392525 2.45505794 0 — 0— E011-T041-S202- 0.994785305 10.03456698 2.467724631 0 −0.99623348 0−0.99623348 E012-T010-S058- 0.994785305 9.914392525 2.451926449 0−0.99623348 0 −0.99623348 E012-T042-S063- 0.994785305 18.987563873.324797258 0 −0.99623348 0 −0.99623348 E013-T041-S104- 0.9947853059.79421807 2.435953353 0 −0.99623348 0 −0.99623348 E013-T070-S199-0.994785305 17.78581932 3.235338653 0 −0.99623348 0 −0.99623348E014-T014-S154- 0.994785305 9.073171341 2.336212574 96.818931635.615808322 0 −0.99623348 E014-T021-S117- 0.994785305 8.7727352042.292528921 0 −0.99623348 0 −0.99623348 E014-T041-S082- 0.9947853058.772735204 2.292528921 0 −0.99623348 0 −0.99623348 E014-T051-S121-0.994785305 7.510903428 2.093078801 4.041777032 1.337698837 0−0.99623348 E011-T062-S062- 0.999110459 14.42093458 2.947460108 0 — 0 —E012-T001-S142- 0.999110459 7.030205609 2.006078739 0 — 0 —E014-T031-S186- 0.999110459 10.69552648 2.548526713 23.944466963.641289747 0 — E014-T056-S103- 0.999110459 10.03456698 2.464599922 0 —0 — E014-T074-S106- 0.999110459 10.5152648 2.526117493 0 — 0 —E012-T028-S115- 1.003435612 8.111775703 2.18525609 0 — 0 —E012-T039-S176- 1.003435612 8.292037385 2.213518814 0 — 0 —E013-T012-S064- 1.003435612 14.66128349 2.966654402 0 — 0 —E013-T019-S116- 1.003435612 6.609595017 1.925343531 29.45598113.92617753 0 — E014-T033-S176- 1.003435612 7.93151402 2.15642861 0 — 0 —E014-T057-X001- 1.003435612 11.41657321 2.631719017 0 — 0 —E014-T061-S186- 1.003435612 0 — 17.57560619 3.212861242 0 —E014-T066-S115- 1.003435612 18.80730218 3.305484345 0 — 0 —E011-T008-S186- 1.007760766 0.060087227 — 19.04534329 3.319607844 0 —E011-T075-S196- 1.007760766 8.772735204 2.283175026 0 — 0 —E011-T082-S142- 1.007760766 8.41221184 2.228946416 0 — 0 —E012-T010-S143- 1.007760766 9.373607479 2.369258406 0 — 0 —E012-T023-S154- 1.007760766 10.21482866 2.481748299 0 — 0 —E012-T045-S136- 1.007760766 9.133258569 2.335438898 0 — 0 —E012-T069-S116- 1.007760766 9.013084114 2.318227124 0 — 0 —E012-T073-S192- 1.007760766 0 — 19.53525566 3.354443628 0 —E013-T031-S176- 1.007760766 11.05604985 2.586098006 0 — 0 —E014-T054-S098- 1.007760766 11.8371838 2.676669461 0 — 0 —E013-T064-S077- 1.012085919 9.013084114 2.315122588 0 — 0 —E014-T019-S100- 1.012085919 10.21482866 2.478643763 0 — 0 —E011-T057-S195- 1.016411073 8.592473522 2.250113096 0 — 0 —E012-T003-S115- 1.016411073 9.433694706 2.371388438 0 — 0 —E012-T059-S193- 1.016411073 9.313520251 2.354675156 0 — 0 —E013-T047-S077- 1.016411073 8.892909659 2.294605121 0 — 0 —E014-T058-S197- 1.016411073 10.99596262 2.572687245 0 — 0 —E011-T025-S100- 1.020736226 7.510903428 2.074431267 0 — 0 —E011-T055-S138- 1.020736226 9.373607479 2.359964767 0 — 0 —E011-T069-S165- 1.020736226 9.493781933 2.376581796 0 — 0 —E012-T021-S175- 1.020736226 8.832822432 2.282724576 19.167821383.319102327 0 — E013-T038-S121- 1.020736226 9.073171341 2.31756504 0 — 0— E013-T060-S083- 1.020736226 7.510903428 2.074431267 0 — 0 —E013-T069-S183- 1.020736226 7.390728973 2.053915141 0 — 0 —E014-T012-S174- 1.020736226 8.712647977 2.26498366 0 — 0 —E014-T028-S069- 1.020736226 8.231950157 2.191754421 0 — 0 —E014-T057-S098- 1.020736226 9.133258569 2.32614526 0 — 0 —E012-T008-S157- 1.02506138 11.47666044 2.623194285 0 — 0 —E013-T005-S098- 1.02506138 9.193345796 2.331590128 0 — 0 —E013-T013-S183- 1.02506138 9.493781933 2.373497174 0 — 0 —E013-T047-S171- 1.02506138 0 — 7.961075972 2.145706333 0 —E014-T037-X001- 1.02506138 8.352124612 2.207328516 0 — 0 —E014-T067-S186- 1.02506138 18.74721496 3.285611656 0 — 0 —E014-T072-S081- 1.02506138 9.734130843 2.406167837 0 — 0 —E011-T039-S100- 1.029386533 9.193345796 2.328512086 0 — 0 —E013-T037-S161- 1.029386533 8.592473522 2.240859199 0 — 0 —E013-T073-S054- 1.029386533 0 — 8.45098834 2.219421529 0 —E013-T075-S058- 1.029386533 13.03892835 2.790317229 0 — 0 —E013-T075-S171- 1.029386533 25.35680997 3.699060185 0 — 0 —E014-T018-S190- 1.029386533 0 — 9.675769259 2.395224446 0 —E014-T036-S169- 1.029386533 9.974479753 2.435036967 0 — 0 —E012-T023-S069- 1.033711687 9.974479753 2.431965478 0 — 0 —E012-T077-X001- 1.033711687 14.48102181 2.928313626 7.1649683752.005332131 0 — E013-T055-S085- 1.033711687 0 — 15.0648053 2.981716424 0— E014-T004-S142- 1.033711687 8.592473522 2.237787711 0 — 0 —E014-T029-S169- 1.033711687 10.03456698 2.439842944 6.8587731461.95018894 0 — E014-T035-S103- 1.033711687 9.193345796 2.325440598 0 — 0— E011-T026-S155- 1.03803684 10.87578816 2.542771229 0 −1.02718013 0−1.02718013 E013-T079-S137- 1.03803684 8.292037385 2.188814828 0−1.02718013 0 −1.02718013 E011-T055-S085- 1.042361994 0 — 14.023741522.878933646 0 — E012-T058-S059- 1.042361994 8.231950157 2.176396839 0 —0 — E013-T047-S130- 1.042361994 0.901308411 1 30.98695725 3.969173262 0— E014-T078-X001- 1.042361994 8.111775703 2.157493638 0 — 0 —E011-T036-S186- 1.046687147 23.07349533 3.556083131 0 — 0 —E013-T037-S154- 1.046687147 8.592473522 2.228612286 0 — 0 —E011-T031-S186- 1.051012301 25.05637384 3.667228274 7.1037293291.982249844 0 — E011-T069-S064- 1.051012301 0 — 13.71754629 2.8431291160 — E014-T071-S059- 1.051012301 8.17186293 2.16087865 0 — 0 —E014-T077-S059- 1.051012301 7.210467291 2.00112819 0 — 0 —E011-T064-S198- 1.055337454 8.592473522 2.222527596 0 — 0 —E012-T009-S176- 1.055337454 8.111775703 2.148356953 0 — 0 —E013-T071-S183- 1.055337454 10.21482866 2.447960393 0 — 0 —E014-T046-S083- 1.055337454 7.93151402 2.119529472 0 — 0 —E011-T026-S085- 1.059662608 7.991601248 2.126170028 0 −1.04240803 0−1.04240803 E012-T026-S143- 1.059662608 7.691165111 2.077141564 0−1.04240803 0 −1.04240803 E013-T058-S104- 1.059662608 0 −1.042408037.777358835 2.091378859 0 −1.04240803 E014-T012-S148- 1.0596626089.914392525 2.4057519 0 −1.04240803 0 −1.04240803 E012-T012-S177-1.063987761 8.472299067 2.198280216 0 — 0 — E012-T023-S085- 1.06831291511.8371838 2.633802369 0 — 0 — E012-T034-S197- 1.068312915 8.3521246122.176839686 0 — 0 — E013-T052-S156- 1.068312915 0 — 91.123700365.477045989 0 — E014-T065-S083- 1.068312915 8.832822432 2.249151123 0 —0 — E011-T034-S077- 1.072638068 8.532386295 2.201369207 0 −1.05146821 0−1.05146821 E012-T078-S120- 1.072638068 8.41221184 2.183065582 0−1.05146821 0 −1.05146821 E013-T015-S099- 1.072638068 8.3521246122.173825944 0 −1.05146821 0 −1.05146821 E013-T026-S104- 1.0726380688.952996887 2.263662783 8.818422616 2.244023057 0 −1.05146821E014-T072-S174- 1.072638068 9.914392525 2.39669172 0 −1.05146821 0−1.05146821 E011-T019-S106- 1.076963222 9.493781933 2.336987141 0−1.05447567 0 −1.05447567 E011-T026-S198- 1.076963222 0 −1.0544756736.00855901 4.155311389 0 −1.05447567 E012-T027-S058- 1.076963222 0−1.05447567 7.593641697 2.048793957 0 −1.05447567 E012-T036-S130-1.076963222 8.231950157 2.152159765 0 −1.05447567 0 −1.05447567E012-T067-S121- 1.076963222 0 −1.05447567 7.654880743 2.059038271 0−1.05447567 E013-T013-S098- 1.081288375 8.352124612 2.16781728 0 — 0 —E013-T045-S137- 1.081288375 9.433694706 2.325701347 0 — 0 —E014-T032-S141- 1.081288375 105.693433 5.679850698 146.48379796.14693579 0 — E011-T001-S165- 1.085613529 0 — 16.77949859 3.091670887 0— E012-T066-S121- 1.085613529 10.09465421 2.411320952 0 — 0 —E014-T032-S199- 1.085613529 8.712647977 2.219392828 8.9409007082.252904729 0 — E011-T007-S193- 1.089938682 10.15474144 2.41612455371.03729329 5.107211455 0 — E011-T058-S084- 1.089938682 9.1933457962.28609515 6.552577916 1.853508548 0 — E011-T080-S175- 1.0899386829.313520251 2.303004323 0 — 0 — E013-T029-S155- 1.089938682 0 —8.573466432 2.195580786 0 — E014-T030-S072- 1.089938682 0 — 63.627368734.950612734 0 — E014-T021-S129- 1.094263836 0 — 30.1296106 3.893772421 0— E011-T001-S102- 1.098588989 11.1762243 2.536575294 7.7161197892.054266383 0 — E011-T003-S130- 1.098588989 9.914392525 2.378740289 0 —0 — E013-T033-S121- 1.098588989 10.15474144 2.410165526 0 — 0 —E013-T062-S175- 1.098588989 9.253433024 2.288615483 0 — 0 —E013-T065-S168- 1.098588989 9.073171341 2.263026413 0 — 0 —E013-T068-X001- 1.098588989 9.493781933 2.322043169 0 — 0 —E014-T035-S135- 1.098588989 8.292037385 2.146575318 0 — 0 —E014-T037-S098- 1.098588989 9.914392525 2.378740289 0 — 0 —E011-T007-S177- 1.102914143 8.652560749 2.19852178 0 — 0 —E011-T057-S186- 1.102914143 9.734130843 2.351743525 0 — 0 —E013-T010-S191- 1.102914143 0 — 7.899836927 2.081388952 0 —E013-T036-S104- 1.102914143 9.253433024 2.285645175 0 — 0 —E014-T013-S169- 1.102914143 9.253433024 2.285645175 0 — 0 —E014-T039-S087- 1.102914143 7.871426793 2.076776203 0 — 0 —E011-T066-S098- 1.107239296 9.133258569 2.265672119 0 — 0 —E011-T069-S120- 1.107239296 10.33500312 2.427358728 0 — 0 —E013-T030-S141- 1.107239296 20.00904673 3.317584643 0 — 0 —E014-T077-S165- 1.107239296 9.553869161 2.324345944 0 — 0 —E012-T059-S168- 1.11156445 10.27491589 2.416732484 0 — 0 —E013-T061-S183- 1.11156445 10.45517757 2.439615636 0 — 0 —E014-T022-S099- 1.11156445 9.253433024 2.279722842 0 — 0 —E014-T040-S161- 1.11156445 17.00468536 3.091988201 225.29845 6.7437706110 — E011-T017-S083- 1.115889603 7.691165111 2.038285237 0 — 0 —E011-T035-S116- 1.115889603 10.27491589 2.413780409 0 — 0 —E011-T065-S063- 1.115889603 8.111775703 2.106467877 0 — 0 —E012-T003-S196- 1.115889603 9.553869161 2.318435742 7.0424902831.926377931 0 — E012-T074-S099- 1.115889603 8.832822432 2.216341233 0 —0 — E013-T004-S109- 1.115889603 8.352124612 2.144029797 0 — 0 —E013-T057-S117- 1.115889603 9.073171341 2.251181698 0 — 0 —E012-T016-S058- 1.120214757 10.33500312 2.41850248 0 — 0 —E012-T021-S174- 1.120214757 0 — 18.8003871 3.223246328 0 —E012-T062-S186- 1.120214757 11.29639876 2.535953548 92.715915565.46601177 0 — E012-T065-S136- 1.120214757 7.991601248 2.084367655 0 — 0— E013-T033-S195- 1.120214757 10.15474144 2.395374765 0 — 0 —E013-T050-S143- 1.120214757 7.751252338 2.045279084 0 — 0 —E013-T054-S054- 1.120214757 11.05604985 2.507474979 0 — 0 —E013-T058-S183- 1.120214757 0 — 8.022315018 2.089287256 0 —E014-T055-S186- 1.120214757 0 — 15.79967385 2.986150917 0 —E014-T066-S058- 1.120214757 0 — 79.61075972 5.248690111 0 —E012-T080-S137- 1.267269976 13.21919003 2.648811192 12.554004422.579691051 0.067975984 — E011-T036-S059- 1.12453991 0 — 7.8998369272.066628456 0 — E012-T006-S195- 1.12453991 8.832822432 2.210455144 0 — 0— E012-T026-S135- 1.12453991 9.013084114 2.236664054 0 — 0 —E014-T047-S098- 1.12453991 27.1594268 3.728395617 0 — 0 —E014-T074-S098- 1.12453991 10.69552648 2.460734457 0 — 0 —E013-T019-S165- 1.128865064 0 — 27.92500495 3.764160792 0 —E014-T030-S195- 1.128865064 9.974479753 2.365996136 0 — 0 —E014-T040-S141- 1.128865064 11.65692212 2.571770203 0 — 0 —E011-T057-S054- 1.133190217 15.20206854 2.925093494 75.017831285.155253345 0 — E013-T038-S189- 1.133190217 0 — 39.62166272 4.2511647710 — E011-T080-S106- 1.137515371 8.772735204 2.192827608 2.8169961130.836502923 0 — E012-T020-S130- 1.137515371 17.90599377 3.144836986 0 —0 — E012-T057-S120- 1.137515371 0 — 8.14479311 2.097015737 0 —E014-T082-S158- 1.137515371 7.570990656 2.00352717 0 — 0 —E012-T058-S174- 1.141840524 8.41221184 2.135682727 0 — 0 —E013-T062-S165- 1.141840524 0 — 12.30904823 2.635484434 0 —E014-T004-S120- 1.141840524 9.854305298 2.341344423 0 — 0 —E014-T035-S142- 1.141840524 10.21482866 2.38848461 0 — 0 —E014-T045-S154- 1.141840524 7.871426793 2.050315087 0 — 0 —E014-T047-S141- 1.141840524 11.23631153 2.514245773 0 — 0 —E011-T059-S189- 1.146165678 10.5152648 2.42371423 0 — 0 —E012-T001-S072- 1.146165678 0 — 10.65559399 2.441189172 0 —E012-T030-S142- 1.146165678 8.111775703 2.085970782 0 — 0 —E013-T009-S102- 1.146165678 9.613956388 2.306129169 0 — 0 —E013-T016-S180- 1.146165678 10.03456698 2.362196659 0 — 0 —E013-T033-S103- 1.146165678 10.21482866 2.385574222 0 — 0 —E013-T067-S145- 1.146165678 9.914392525 2.346398478 0 — 0 —E014-T018-S195- 1.150490831 7.691165111 2.014883613 0 −1.10466598 0−1.10466598 E014-T076-S106- 1.150490831 9.433694706 2.27851224 0−1.10466598 0 −1.10466598 E012-T037-S194- 1.154815985 7.6911651112.011984921 0 — 0 — E012-T046-S141- 1.154815985 20.3695701 3.309921307 0— 0 — E013-T012-S121- 1.154815985 8.472299067 2.136149959 0 — 0 —E013-T017-S147- 1.154815985 9.313520251 2.258900266 0 — 0 —E013-T053-S183- 1.154815985 10.81570094 2.455068639 0 — 0 —E013-T073-S154- 1.159141138 9.013084114 2.213356948 0 — 0 —E014-T051-S155- 1.159141138 8.41221184 2.124076239 0 — 0 —E011-T015-S081- 1.163466292 8.111775703 2.074387591 0 — 0 —E011-T047-S085- 1.163466292 8.712647977 2.166520031 0 — 0 —E012-T024-S165- 1.163466292 9.253433024 2.244690482 0 — 0 —E013-T002-S169- 1.163466292 19.10773832 3.216334271 0 — 0 —E013-T058-S141- 1.163466292 17.66564486 3.108968804 0 — 0 —E014-T021-S084- 1.167791445 9.133258569 2.224800306 0 — 0 —E011-T010-S130- 1.172116599 9.79421807 2.313085285 0 — 0 —E011-T076-S171- 1.172116599 9.433694706 2.264076671 0 — 0 —E012-T042-S106- 1.172116599 7.871426793 2.030064603 0 — 0 —E013-T006-S191- 1.172116599 0 — 29.63969824 3.818228635 0 —E013-T007-S143- 1.172116599 19.82878505 3.261405235 0 — 0 —E014-T009-S063- 1.172116599 9.493781933 2.272361262 0 — 0 —E011-T025-S169- 1.176441752 9.253433024 2.236063714 0 −1.12197141 0−1.12197141 E013-T082-S199- 1.176441752 10.15474144 2.357613757 0−1.12197141 0 −1.12197141 E012-T021-S099- 1.180766906 8.2319501572.081799861 0 — 0 — E012-T042-S069- 1.180766906 8.532386295 2.1280018430 — 0 — E013-T056-S115- 1.180766906 8.892909659 2.181559329 0 — 0 —E013-T079-S169- 1.180766906 0 — 22.41349081 3.424432569 0 —E014-T038-S189- 1.180766906 8.892909659 2.181559329 0 — 0 —E014-T063-S137- 1.180766906 10.99596262 2.459641453 0 — 0 —E011-T044-S121- 1.185092059 8.952996887 2.18743693 0 — 0 —E012-T073-S180- 1.185092059 9.373607479 2.247151719 0 — 0 —E012-T029-S175- 1.189417213 9.313520251 2.235918039 0 — 0 —E012-T051-S098- 1.189417213 10.87578816 2.43940446 0 — 0 —E011-T028-S168- 1.193742366 15.32224299 2.895373315 0 — 0 —E011-T071-S120- 1.193742366 0 — 22.47472986 3.419642546 0 —E013-T032-S126- 1.193742366 9.674043616 2.282640801 0 — 0 —E013-T070-S085- 1.193742366 10.21482866 2.353941569 0 — 0 —E013-T072-S115- 1.193742366 8.111775703 2.054338129 0 — 0 —E012-T020-S062- 1.19806752 9.734130843 2.287897771 0 — 0 —E013-T079-S121- 1.19806752 9.253433024 2.221799421 0 — 0 —E014-T014-S143- 1.202392673 26.37829284 3.63588887 0 — 0 —E013-T050-S169- 1.206717827 9.193345796 2.207653601 0 — 0 —E013-T021-S136- 1.21104298 14.84154517 2.840914087 0 −1.14472707 0−1.14472707 E013-T082-S161- 1.21104298 10.33500312 2.357985813 0−1.14472707 0 −1.14472707 E014-T021-S154- 1.21104298 9.0731713412.187718984 0 −1.14472707 0 −1.14472707 E014-T028-S165- 1.21104298 0−1.14472707 34.7837781 4.016506742 0 −1.14472707 E014-T029-S117-1.21104298 0 −1.14472707 43.96963499 4.346152199 0 −1.14472707E011-T027-S176- 1.215368134 9.553869161 2.252153643 0 — 0 —E013-T029-S154- 1.215368134 9.253433024 2.210488669 0 — 0 —E013-T070-S170- 1.215368134 9.013084114 2.176268044 0 — 0 —E011-T080-X001- 1.219693287 10.39509034 2.359980116 0 — 0 —E012-T007-S083- 1.219693287 10.09465421 2.321432456 45.745567324.39639732 0 — E013-T044-S082- 1.219693287 10.03456698 2.31359777 0 — 0— E014-T067-S085- 1.219693287 10.5152648 2.37511534 4.1642551241.218199933 0 — E012-T045-S115- 1.224018441 19.34808723 3.193652529 0−1.15316875 0 −1.15316875 E013-T044-S137- 1.224018441 9.9744797532.302911895 0 −1.15316875 0 −1.15316875 E013-T070-S176- 1.2240184419.974479753 2.302911895 0 −1.15316875 0 −1.15316875 E014-T017-S116-1.224018441 8.472299067 2.090545881 0 −1.15316875 0 −1.15316875E011-T044-S189- 1.228343594 9.854305298 2.284223785 0 — 0 —E012-T050-S109- 1.228343594 19.34808723 3.190849576 0 — 0 —E013-T039-S104- 1.228343594 9.253433024 2.202063422 0 — 0 —E011-T030-S106- 1.232668748 10.27491589 2.336275546 0 −1.15876922 0−1.15876922 E013-T073-S058- 1.232668748 0 −1.15876922 29.945893473.792906851 0 −1.15876922 E014-T039-S105- 1.232668748 11.416573212.475425941 0 −1.15876922 0 −1.15876922 E012-T057-S194- 1.23699390110.69552648 2.386323579 0 — 0 — E012-T065-S077- 1.236993901 11.476660442.479598599 0 — 0 — E011-T072-S197- 1.241319055 8.111775703 2.0233842010 — 0 — E013-T057-S137- 1.241319055 0 — 15.55471767 2.884822469 0 —E013-T060-S194- 1.249969362 9.013084114 2.153909144 0 — 0 —E012-T081-S064- 1.254294515 26.31820561 3.599114808 0 — 0 —E011-T016-S083- 1.258619669 9.313520251 2.191023584 0 — 0 —E012-T033-S143- 1.262944822 10.93587539 2.399031067 0 — 0 —E014-T045-S100- 1.262944822 17.18494704 3.006471412 0 — 0 —E014-T074-S100- 1.262944822 8.472299067 2.065513224 0 — 0 —E014-T001-S170- 1.267269976 9.734130843 2.243177283 0 — 0 —E012-T075-S059- 1.271595129 10.15474144 2.295879444 0 — 0 —E012-T066-S186- 1.275920283 10.15474144 2.293135141 0 — 0 —E013-T029-S141- 1.275920283 12.73849221 2.593701747 0 — 0 —E013-T014-S143- 1.28457059 0 −1.19192302 11.57417968 2.460469359 0−1.19192302 E012-T050-S177- 1.288895743 10.93587539 2.382580723 0 — 0 —E014-T044-S195- 1.288895743 0 — 53.27796997 4.567643108 0 —E014-T064-S083- 1.288895743 8.952996887 2.120479241 0 — 0 —E014-T063-S142- 1.29754605 8.712647977 2.079770896 0 — 0 —E011-T005-S064- 1.301871204 8.712647977 2.077057561 0 — 0 —E013-T051-S106- 1.301871204 10.57535203 2.330177051 7.4711636051.87975304 0 — E012-T033-S102- 1.353773046 8.832822432 2.06263037 0−1.23497522 0 −1.23497522 E011-T048-S072- 1.362423353 9.1332585692.100758751 0 — 0 — E011-T070-S059- 1.37107366 9.974479753 2.210540163 0— 0 — E012-T082-S177- 1.37107366 8.832822432 2.052065108 0 — 0 —E013-T082-S100- 1.37107366 21.63140187 3.254713566 0 — 0 —E013-T038-S126- 1.379723967 21.45114019 3.23792257 0 −1.25079424 0−1.25079424 E013-T046-S098- 1.379723967 8.892909659 2.055600663 0−1.25079424 0 −1.25079424 E014-T072-S186- 1.379723967 12.377968852.490992946 0 −1.25079424 0 −1.25079424 E011-T066-S085- 1.3926994278.472299067 1.985075456 162.2834717 6.092595777 0 — E011-T078-S082-1.401349734 10.09465421 2.207947264 0 — 0 — E014-T026-S121- 1.40134973410.03456698 2.200112577 0 — 0 — E013-T045-S176- 1.410000041 16.6441622.872085835 7.042490283 1.738609117 0 — E014-T054-S145- 1.4143251959.79421807 2.160566822 0 — 0 — E013-T039-S116- 1.431625809 9.7341308432.142212237 0 — 0 — E013-T004-S177- 1.448926423 9.734130843 2.1319840450 — 0 — E011-T057-S104- 1.45757673 9.553869161 2.102463637 0 — 0 —E013-T021-S083- 1.461901883 18.92747664 3.016913866 3.3681475270.827248316 0 — E013-T054-S116- 1.461901883 16.52398754 2.831485923 0 —0 — E013-T013-S142- 1.47055219 9.493781933 2.086629277 0 — 0 —E013-T050-X001- 1.47055219 12.55823053 2.456263468 0 — 0 —E014-T059-S186- 1.474877344 0 — 15.92215194 2.773484112 0 —E011-T040-S154- 1.479202497 9.734130843 2.114257361 0 — 0 —E012-T019-S058- 1.487852804 9.193345796 2.034654635 0 −1.31490113 0−1.31490113 E014-T077-S183- 1.505153418 20.00904673 3.06803983912.61524346 2.442251917 0 — E012-T022-S069- 1.509478572 10.154741442.15219754 0 — 0 — E014-T013-S083- 1.509478572 9.854305298 2.112807861 0— 0 — E013-T038-S100- 1.513803725 11.41657321 2.304323151 0 −1.329872010 −1.32987201 E012-T011-S202- 1.522454032 11.1762243 2.271166957 0 — 0 —E013-T046-S168- 1.522454032 0 — 14.2686977 2.597677133 0 —E013-T072-S098- 1.526779186 38.03521496 3.949404716 265.10382996.718545871 0 — E014-T055-S115- 1.526779186 10.99596262 2.247177433 0 —0 — E013-T002-S102- 1.535429493 9.493781933 2.049232655 0 — 0 —E014-T003-S143- 1.535429493 6.429333335 1.551002602 67.301711494.751619669 0 — E014-T061-S109- 1.539754646 9.914392525 2.103470798 0 —0 — E012-T047-S064- 1.5440798 9.974479753 2.108936721 0 — 0 —E014-T019-S063- 1.55705526 18.44677882 2.926975962 0 — 0 —E011-T002-S147- 1.570030721 19.10773832 2.967893309 0 — 0 —E013-T065-S183- 1.574355874 11.29639876 2.255952448 0 — 0 —E013-T017-S165- 1.583006181 5.107414331 1.241510626 146.30008085.833563315 0 — E014-T070-S109- 1.608957102 19.88887228 3.00118948 0 — 0— E011-T044-X001- 1.613282256 17.54547041 2.827132001 0 — 0 —E013-T017-S109- 1.613282256 14.96171963 2.610681227 0 — 0 —E011-T037-S177- 1.617607409 9.914392525 2.059911193 0 — 0 —E013-T007-S175- 1.617607409 18.08625546 2.866213445 0 — 0 —E011-T030-S138- 1.621932563 9.613956388 2.017260042 0 — 0 —E011-T038-S157- 1.64355833 18.80730218 2.905479329 0 −1.40248116 0−1.40248116 E012-T030-S143- 1.647883484 14.2406729 2.525015057 0−1.40483964 0 −1.40483964 E011-T060-S186- 1.660858944 11.056049852.179793349 0 — 0 — E013-T069-S137- 1.691135019 10.39509034 2.0821256830 — 0 — E013-T057-S072- 1.695460172 10.09465421 2.041261205 0 — 0 —E013-T033-S197- 1.71708594 13.15910281 2.381597749 0 — 0 —E014-T070-S135- 1.721411093 18.32660437 2.828161372 0 — 0 —E014-T068-S186- 1.751687168 19.04765109 2.865044843 0 — 0 —E013-T028-S115- 1.756012321 30.04361371 3.493642265 0 — 0 —E013-T016-S109- 1.760337475 17.54547041 2.748150298 0 −1.46484466 0−1.46484466 E011-T030-S104- 1.764662628 14.60119626 2.496481312 0 — 0 —E012-T013-S069- 1.768987782 10.21482866 2.017976987 0 — 0 —E012-T069-S154- 1.768987782 10.15474144 2.01022648 0 — 0 —E013-T068-S102- 1.794938703 11.29639876 2.137347308 0 — 0 —E014-T020-S142- 1.799263856 14.30076013 2.450483941 0 — 0 —E013-T062-S186- 1.993895764 32.92780063 3.502372012 33.130323853.510958206 0.067975984 — E014-T002-S104- 1.80358901 20.429657322.934262001 0 — 0 — E011-T056-S129- 1.833865084 0 — 82.060321564.873316476 0 — E014-T067-S054- 1.833865084 0 — 12.49276537 2.2513430820 — E014-T006-S141- 1.838190238 15.74285359 2.560502238 0 — 0 —E013-T020-S117- 1.864141159 0 — 40.35653128 3.851940684 0 —E012-T080-S142- 1.868466312 10.63543925 2.020174196 0 — 0 —E014-T073-S145- 1.868466312 17.966081 2.725070123 0 — 0 —E013-T019-S101- 1.872791466 0 — 30.74200106 3.465867903 0 —E014-T022-S186- 1.89009208 0 — 18.31047474 2.740196269 0 —E013-T028-S195- 1.898742387 14.84154517 2.450214031 0 — 0 —E012-T042-S194- 1.907392694 10.75561371 2.015552007 0 — 0 —E012-T057-S082- 1.911717847 17.84590655 2.694308728 7.5324026511.551081488 0 — E012-T017-S186- 1.920368154 0 — 22.84216414 3.0292930360 — E012-T047-S176- 1.929018461 13.69988785 2.327315957 0 — 0 —E014-T028-S138- 1.929018461 20.48974455 2.875159142 0 — 0 —E012-T017-S168- 1.941993922 10.99596262 2.027682761 0 — 0 —E014-T037-S121- 1.954969382 27.33968848 3.261608812 16.718259542.584021808 0 — E013-T029-S145- 1.972269996 0 — 36.74342757 3.6665883640 — E013-T063-S175- 1.972269996 11.05604985 2.020120208 0 — 0 —E014-T061-S116- 1.998220917 18.6270405 2.710664056 0 — 0 —E012-T036-S183- 2.006871224 19.46826169 2.767053596 0 — 0 —E014-T011-S177- 2.045797606 18.38669159 2.670174641 0 — 0 —E011-T045-S174- 2.050122759 17.48538318 2.599445736 0 — 0 —E013-T045-S186- 2.050122759 0 — 59.34063552 4.306190678 0 —E014-T019-S177- 2.06309822 21.09061683 2.850370169 0 — 0 —E014-T052-S165- 2.071748527 0 — 17.08569382 2.557716927 0 —E014-T074-S169- 2.071748527 16.76433645 2.531851783 0 — 0 —E014-T045-S085- 2.07607368 13.94023676 2.280041046 0 −1.62109006 0−1.62109006 E012-T024-S157- 2.080398834 13.51962617 2.236815249 0 — 0 —E014-T079-S098- 2.080398834 17.84590655 2.613062134 0 — 0 —E012-T001-S168- 2.110674908 12.73849221 2.142924144 0 — 0 —E014-T019-S121- 2.110674908 17.66564486 2.585085818 0 — 0 —E012-T006-S186- 2.115000062 0 — 25.23048693 3.073940492 0 —E013-T030-S192- 2.115000062 18.68712773 2.659948546 0 — 0 —E013-T046-S120- 2.115000062 11.47666044 2.00192773 0 — 0 —E013-T001-S190- 2.119325215 76.43095329 4.633604525 52.910535694.111261367 0 — E013-T064-S115- 2.119325215 21.45114019 2.8474828362.449561838 0.145179149 0 — E013-T011-S141- 2.127975522 23.854629292.990213457 66.81179912 4.438235193 0 — E012-T001-S177- 2.1496012937.91504051 3.627086745 0 — 0 — E013-T045-S146- 2.171227057 15.98320252.420995449 0 — 0 — E012-T014-S058- 2.179877364 15.38233022 2.3650975480 — 0 — E012-T032-S186- 2.188527671 5.227588786 0.965783283 11.941613962.021055242 0 — E011-T072-S199- 2.192852825 12.85866667 2.117870505 0 —0 — E012-T069-X001- 2.192852825 29.92343926 3.275782824 0 — 0 —E013-T017-S115- 2.197177978 11.89727103 2.012194872 0 — 0 —E014-T044-S102- 2.210153439 15.32224299 2.346125163 0 — 0 —E013-T027-S072- 2.218803746 0 — 33.49775813 3.421906088 0 —E014-T010-S084- 2.231779206 18.74721496 2.611248655 0 — 0 —E013-T046-S064- 2.240429513 9.613956388 1.711705568 16.595781452.44097263 0 — E014-T047-S177- 2.24907982 19.40817446 2.651044045 0 — 0— E014-T055-S059- 2.257730127 12.43805608 2.044385456 0 — 0 —E011-T003-S169- 2.262055281 18.38669159 2.571213487 0 — 0 —E012-T024-S085- 2.266380434 18.92747664 2.608994301 0 — 0 —E014-T029-S137- 2.275030741 14.36084735 2.229677448 0 — 0 —E011-T019-S186- 2.292331355 0 — 102.636641 4.976280809 0 —E014-T009-S183- 2.300981662 18.26651714 2.545128766 0 — 0 —E014-T021-S077- 2.300981662 17.12485982 2.457002808 0 — 0 —E013-T033-S137- 2.305306816 19.82878505 2.655722587 0 — 0 —E014-T048-S115- 2.305306816 18.74721496 2.578793096 0 — 0 —E013-T055-S177- 2.32260743 18.50686605 2.553594263 0 — 0 —E013-T013-S109- 2.326932583 18.98756387 2.586838107 0 — 0 —E012-T069-S168- 2.331257737 13.39945172 2.111874996 0 — 0 —E012-T006-S085- 2.33558289 0 — 17.75932332 2.49159699 0 —E012-T069-S143- 2.339908044 21.0305296 2.72162389 0 — 0 —E014-T072-S143- 2.348558351 30.46422431 3.232100363 0 — 0 —E014-T072-S142- 2.357208658 31.84623054 3.290393703 0 — 0 —E013-T012-S137- 2.365858965 41.88079752 3.671285106 0 — 0 —E014-T014-S168- 2.391809886 0 — 58.17709365 4.124911631 0 —E014-T016-S054- 2.426411114 13.5797134 2.089192193 15.126044352.234622428 0 — E012-T055-S054- 2.443711728 22.53271028 2.772631201120.2122472 5.137427284 0 −1.78396438 E013-T019-S170- 2.48696326318.32660437 2.470545112 0 — 0 — E014-T015-S180- 2.491288416 17.605557642.413902182 0 — 0 — E013-T057-S077- 2.504263877 20.79018069 2.6364948380 — 0 — E011-T019-S105- 2.530214798 18.20642991 2.443761503 15.309761492.207907811 0 — E013-T047-S186- 2.538865105 15.02180686 2.1786781826.981251237 1.173328169 0 −1.82328677 E013-T048-S098- 2.56 19.23 2.5149.24 3.82 0.00 −1.83 E011-T019-S059- 2.56 13.82 2.06 0.00 −1.83 0.00−1.83 E013-T065-S084- 2.57 16.34 2.28 0.00 −1.84 0.00 −1.84E011-T029-S143- 2.60 16.16 2.25 0.00 −1.85 0.00 −1.85 E014-T044-S175-2.62 13.58 2.01 0.00 −1.86 0.00 −1.86 E013-T030-S101- 2.63 0.00 −1.8651.26 3.85 0.00 −1.86 E013-T027-S186- 2.65 8.71 1.41 98.90 4.78 0.00−1.87 E013-T001-X001- 2.68 20.13 2.52 0.00 −1.88 0.00 −1.88E014-T047-S186- 2.69 18.93 2.43 82.67 4.50 0.00 −1.88 E012-T026-S180-2.71 14.84 2.09 0.00 −1.89 0.00 −1.89 E014-T081-S102- 2.73 16.22 2.210.00 −1.90 0.00 −1.90 E014-T035-S199- 2.74 0.00 −1.90 30.19 3.06 0.00−1.90 E012-T029-S157- 2.75 14.60 2.06 0.00 −1.91 0.00 −1.91E012-T020-S156- 2.75512278 17.18494704 2.27581274 0 −1.90886008 0−1.90886008 E012-T051-S116- 2.798374315 15.02180686 2.076582867 0 — 0 —E012-T057-S143- 2.811349775 14.42093458 2.016516284 0 — 0 —E013-T005-S059- 2.832975543 10.81570094 1.624168518 15.922151942.142376346 0 — E012-T039-S126- 2.837300696 0 — 44.21459117 3.5586246930 — E012-T070-S130- 2.837300696 19.40817446 2.410983411 0 — 0 —E013-T016-S136- 2.850276157 18.02616823 2.304951209 0 — 0 —E013-T032-S087- 2.85460131 0 — 21.43366608 2.541011851 0 —E012-T031-S116- 2.876227078 18.50686605 2.33125702 0 — 0 —E012-T078-S069- 2.880552231 19.22791278 2.382013584 0 — 0 —E013-T021-S137- 2.884877385 18.38669159 2.319125654 0 — 0 —E012-T001-S120- 2.902177999 17.90599377 2.276492192 0 — 0 —E013-T063-S130- 2.915153459 19.40817446 2.382006375 0 — 0 —E013-T046-S195- 2.932454073 25.71733334 2.764274198 0 — 0 —E012-T016-S186- 2.936779227 20.73009346 2.464606666 0 — 0 —E014-T044-S158- 2.954079841 0 — 76.97748075 4.301643638 0 —E013-T056-X001- 2.997331376 19.34808723 2.347784103 0 — 0 —E014-T077-S085- 3.001656529 0 — 36.80466661 3.239895084 0 —E013-T046-X001- 3.01463199 0 — 20.69879753 2.434275445 0 —E013-T071-S085- 3.01463199 20.54983178 2.424336956 0 — 0 —E013-T019-X001- 3.023282297 18.56695327 2.28197426 0 — 0 —E014-T073-S137- 3.031932604 18.02616823 2.238441611 0 — 0 —E014-T057-S121- 3.036257757 15.80294081 2.057623539 0 — 0 —E014-T030-S145- 3.044908064 16.94459813 2.149370797 0 — 0 —E014-T045-S186- 3.049233218 14.48102181 1.934780055 479.37925166.890381292 0 — E012-T075-S137- 3.057883525 20.30948287 2.392696222 0 —0 — E012-T030-S199- 3.079509292 20.12922119 2.372772059 0 — 0 —E012-T002-S143- 3.088159599 42.12114643 3.39887211 20.270124212.379305033 0 — E014-T014-S193- 3.109785367 16.70424922 2.106960709 0 —0 — E012-T078-S137- 3.127085981 18.80730218 2.262836994 0 — 0 —E014-T032-S109- 3.127085981 19.10773832 2.284555419 0 — 0 —E014-T002-S194- 3.131411134 18.32660437 2.225881641 0 — 0 —E012-T020-S109- 3.140061441 21.15070405 2.419628472 0 — 0 —E013-T020-S186- 3.153036902 16.88451091 2.106472069 95.226716444.534198904 0 — E014-T042-S186- 3.161687209 0 — 21.67862226 2.4460925570 — E012-T006-S121- 3.166012362 9.974479753 1.397413525 41.887507423.363818445 0 — E014-T077-S186- 3.191963283 18.92747664 2.24906105254.44151184 3.725268613 0 — E014-T049-S137- 3.222239358 18.326604372.19450791 0 — 0 — E013-T061-S195- 3.226564512 0 −2.07948547 21.801100362.431546073 0 −2.07948547 E013-T038-S183- 3.235214819 0 — 29.823415372.863519675 0 — E013-T007-S058- 3.239539972 0 — 35.51864665 3.1066536680 — E014-T030-X001- 3.26116574 13.45953894 1.762701479 68.587731464.029512905 0 — E014-T034-S186- 3.265490893 0.180261682 −1.85360502315.0748914 6.21141084 0 — E011-T013-S177- 3.278466354 19.468261692.258222933 0 — 0 — E014-T034-S109- 3.300092121 20.85026792 2.3452114970 — 0 — E012-T006-X001- 3.304417275 17.18494704 2.078854878 0 — 0 —E013-T080-S183- 3.304417275 13.94023676 1.795313165 16.289586222.006013496 0 — E014-T035-S109- 3.304417275 0 — 49.48114912 3.5518549050 — E012-T047-S121- 3.321717889 20.3695701 2.30588108 7.0424902830.896037388 0 −2.1116049 E012-T014-S083- 3.343343656 19.107738322.210872807 0 — 0 — E014-T023-S136- 3.34766881 17.72573209 2.106708172 0— 0 — E014-T073-S058- 3.34766881 22.23227415 2.417816433 0 — 0 —E012-T074-S121- 3.382270038 20.18930842 2.27358621 0 — 0 —E013-T073-S115- 3.386595191 27.09933957 2.679362741 5.2053189050.50040378 0 — E012-T052-S117- 3.442822187 21.0305296 2.30995587 0 — 0 —E014-T017-X001- 3.53365041 24.27523988 2.47897973 0 — 0 —E013-T062-S195- 3.568251638 8.592473522 1.070260755 22.842164142.383801167 0 — E011-T077-S186- 3.633128941 27.70021184 2.631002647126.2749127 4.779817433 0 — E012-T030-S135- 3.641779248 21.210791282.258510973 0 — 0 — E011-T022-S202- 3.654754708 20.42965732 2.2028317310 — 0 — E013-T014-X001- 3.663405015 19.22791278 2.116891825 0 — 0 —E013-T056-S104- 3.732607471 19.82878505 2.137871516 11.880374911.444467414 0 — E013-T028-S186- 3.771533853 21.99192524 2.268602263173.7351733 5.194573127 0 — E011-T062-S186- 3.775859006 68.980137093.87311334 333.262888 6.129079131 0 — E013-T015-S104- 3.7758590067.030205609 0.749676682 56.58487845 3.591857865 0 — E014-T060-S168-3.806135081 19.16782555 2.069106442 0 — 0 — E014-T014-S142- 3.8493866160 — 58.36081078 3.613636615 0 — E013-T010-S137- 3.901288458 18.62704052.001609688 0 — 0 — E014-T002-S186- 3.909938765 4.806978194 0.24208258563.4436516 3.714261307 0 — E014-T057-S058- 3.909938765 19.588436142.068057313 0 — 0 — E014-T019-X001- 3.922914225 25.05637384 2.40405181610.96178922 1.280848694 0 — E013-T029-S126- 3.9531903 19.588436142.055404295 0 −2.30835805 0 −2.30835805 E014-T073-S062- 3.9661657635.57163864 2.880520948 0 — 0 — E011-T077-S138- 3.974816067 10.214828661.172692488 63.4436516 3.695323153 0 — E011-T073-S104- 3.98779152821.0305296 2.143031105 6.613816962 0.610218721 0 — E012-T011-S183-4.005092142 20.54983178 2.106208074 0 — 0 — E011-T009-S138- 4.0353682169.493781933 1.05936553 66.3831258 3.742218169 0 — E013-T051-S126-4.095920365 25.65724611 2.38711311 0 — 0 — E014-T044-S141- 4.199724049 0— 22.84216414 2.197008228 0 — E013-T065-S100- 4.29920258 0 −2.4057752862.15763163 3.575109887 0 −2.40577528 E012-T005-S177- 4.662515474 0 —29.76217633 2.44164258 0 — E012-T057-S069- 4.666840627 12.017445491.199829831 98.59486397 4.135454811 0 — E013-T006-S062- 4.80957069326.25811839 2.230182515 0 — 0 — E012-T032-S054- 4.935000144 0 —32.33421626 2.489683837 0 — E014-T026-S102- 5.133957205 28.301084122.256064038 0 — 0 — E013-T007-S176- 5.172883587 0 — 55.543814673.195352727 0 — E013-T022-S138- 5.198834508 49.93248599 3.038517243300.4387594 5.603724134 0 — E013-T020-S157- 5.306963345 28.361171352.218892071 0 — 0 — E012-T028-S199- 5.916809989 8.832822432 0.50749876531.41563057 2.228510907 0 — E014-T073-S192- 6.037914287 9.5538691610.584552154 36.9271447 2.430010915 0 — E014-T042-S177- 6.12874251440.9200749 5.953994047 52.48186237 2.907330188 0 — E012-T061-S186-6.206595273 8.892909659 0.457077077 54.13531661 2.935586995 0 —E012-T076-S168- 6.414202641 24.27523988 1.769361333 102.14672863.798262709 0 — E014-T057-S135- 6.427178102 30.76466044 2.0965365186.246382686 — 0 — E012-T046-S186- 6.578558475 0 — 125.96871754.066405822 0 — E014-T068-S143- 6.600184242 29.56291589 2.007675894 0 —0 — E012-T077-S186- 6.816441917 9.974479753 0.48956861 262.5930295.075656372 0 — E014-T029-S186- 7.019724132 15.50250468 1.041060491105.5761152 3.732187731 0 — E014-T012-S176- 7.599294701 0 — 36.498471382.124541543 0 — E012-T021-S186- 8.261043186 19.10773832 1.118504203125.7850004 3.775065552 0 — E013-T021-S186- 8.745460378 9.0731713410.047715714 84.08121008 3.126038307 0 −3.28473034 E013-T057-S186-9.57588985 28.18090966 1.464245801 135.5832477 3.690929583 0 —E014-T017-S186- 15.04720903 87.06639254 2.456269168 261.85816044.033890217 0 —

TABLE 19 Constructs with Particularly Noteworthy Enrichments inLibraries 1A and 1.1A. P3 P4 Contains Contains Cytokine ITAM CytokineITAM BlockSequence Gene receptor? motif? Gene receptor? motif?M001-S116-S044 IL6R Y N CD8B N N M024-S192-S045 MYD88 N N CD8B N NM001-S047-S102 CD27 Y N IL1RL1 Y N M048-S195-S043 MYD88 N N CD8A N NM012-S216-S211 TNFRSF18 Y N TNFRSF4 Y N M030-S170-S194 IL31RA Y N MYD88N N

TABLE 20 Constructs with Particularly Noteworthy Enrichments inLibraries 2B and 2.1B. P3 P4 Contains Contains Cytokine ITAM CytokineITAM BlockSequence Gene receptor? motif? Gene receptor? motif?M007-S049-S051 CD28 N N CD40 Y N M007-S050-S039 CD40 Y N CD3G N YM012-S050-S043 CD40 Y N CD8A N N M012-S161-S213 IL22RA1 Y N TNFRSF9 Y NM030-S142-S049 IL13RA2 Y N CD28 N N M001-S145-S130 IL17RB Y N IL10RB Y NM018-S085-S039 IFNGR2 Y N CD3G N Y M018-S075-S053 FCGR2C Y Y CD79B N YM012-S135-S074 IL11RA Y N FCER1G Y Y M007-S214-S077 TNFRSF14 Y N GHR Y N

TABLE 21 Constructs with Particularly Noteworthy Enrichments inLibraries 3A and 3.1A. P3 P4 Contains Contains Cytokine ITAM CytokineITAM BlockSequence Gene receptor? motif? Gene receptor? motif?E008/E013-T041-S186-S050 MPL Y N CD40 Y N E006/E011-T077-S186-S211 MPL YN TNFRSF4 Y N E007/E012-T021-S186-S051 MPL Y N CD40 Y NE009/E014-T041-S186-S053 MPL Y N CD79B N Y E007/E012-T073-S186-S053 MPLY N CD79B N Y E006/E011-T017-S186-S051 MPL Y N CD40 Y NE006/E011-T031-S186-S211 MPL Y N TNFRSF4 Y N E006/E011-T011-S186-S050MPL Y N CD40 Y N E006/E011-T011-S186-S047 MPL Y N CD27 Y NE007/E012-T001-S186-S050 MPL Y N CD40 Y N E006/E011-T041-S186-S051 MPL YN CD40 Y N E008/E013-T028-S186-S076 MPL Y N FCGR2A Y YE009/E014-T029-S199-S053 OSMR Y N CD79B N Y E009/E014-T062-S186-S216 MPLY N TNFRSF18 Y N E007/E012-T006-S058-S051 CSF2RA Y N CD40 Y NE009/E014-T076-S186-S211 MPL Y N TNFRSF4 Y N E007/E012-T001-S186-S047MPL Y N CD27 Y N

TABLE 22 Constructs with Particularly Noteworthy Enrichments inLibraries 3B and 3.1B. P3 P4 Contains Contains Cytokine ITAM CytokineITAM BlockSequence Gene receptor? motif? Gene receptor? motif?E007/E012-T017-S186-S051 MPL Y N CD40 Y N E007/E012-T073-S186-S053 MPL YN CD79B N Y E008/E013-T028-S186-S047 MPL Y N CD27 Y NE006/E011-T011-S186-S047 MPL Y N CD27 Y N E007/E012-T082-S176-S214 LEPRY N TNFRSF14 Y N E006/E011-T046-S186-S052 MPL Y N CD79A N YE008/E013-T029-S186-S052 MPL Y N CD79A N Y E009/E014-T011-S186-S053 MPLY N CD79B N Y E008/E013-T032-S186-S039 MPL Y N CD3G N YE007/E012-T034-S186-S051 MPL Y N CD40 Y N E007/E012-T041-S192-S213 MYD88N N TNFRSF9 Y N E006/E011-T014-S069-S213 EPOR Y N TNFRSF9 Y NE006/E011-T022-S186-S053 MPL Y N CD79B N Y E006/E011-T023-S115-S075IL5RA Y N FCGR2C Y Y E006/E011-T029-S106-S213 IL2RG Y N TNFRSF9 Y NE006/E011-T032-S155-S080 IL18RAP Y N ICOS N N E006/E011-T041-S186-S216MPL Y N TNFRSF18 Y N E006/E011-T057-S135-S080 IL11RA Y N ICOS N NE006/E011-T072-S191-X002 MYD88 N N N/A N N E006/E011-T077-S186-S216 MPLY N TNFRSF18 Y N E006/E011-T080-S141-S080 IL13RA1 Y N ICOS N NE007/E012-T001-X001-S214 N/A N N TNFRSF14 Y N E007/E012-T007-S059-S211CSF2RA Y N TNFRSF4 Y N E007/E012-T016-S186-S052 MPL Y N CD79A N YE007/E012-T031-S186-S053 MPL Y N CD79B N Y E007/E012-T044-S102-S052IL1RL1 Y N CD79A N Y E007/E012-T044-S142-X002 IL13RA2 Y N N/A N/A N/AE007/E012-T055-S069-S053 EPOR Y N CD79B N Y E007/E012-T063-S176-S216LEPR Y N TNFRSF18 Y N E007/E012-T065-S157-S075 IL20RB Y N FCGR2C Y YE008/E013-T008-S085-X002 IFNGR2 Y N N/A N/A N/A E008/E013-T011-S085-S048IFNGR2 Y N CD28 N N E008/E013-T021-S109-X002 IL 3 RA Y N N/A N/A N/AE008/E013-T021-S168-S211 IL27RA Y N TNFRSF4 Y N E008/E013-T032-S064-S214CSF3R Y N TNFRSF14 Y N E008/E013-T037-S170-S215 IL31RA Y N TNFRSF18 Y NE008/E013-T038-S176-S048 LEPR Y N CD28 N N E008/E013-T039-S137-S216IL12RB1 Y N TNFRSF18 Y N E008/E013-T041-S141-S053 IL13RA1 Y N CD79B N YE008/E013-T045-S177-S048 LEPR Y N CD28 N N E008/E013-T048-S109-S074IL3RA Y N FCER1G Y Y E008/E013-T073-S199-S075 OSMR Y N FCGR2C Y YE009/E014-T001-S157-S074 IL20RB Y N FCER1G Y Y E009/E014-T005-S196-S049MYD88 N N CD28 N N E009/E014-T011-S130-X002 IL10RB Y N N/A N/A N/AE009/E014-T013-S155-X002 IL18RAP Y N N/A N/A N/AE009/E014-T017-S186-S076 MPL Y N FCGR2A Y Y E009/E014-T021-S142-S080IL13RA2 Y N ICOS N N E009/E014-T023-S082-S076 IFNAR2 Y N FCGR2A Y YE009/E014-T038-S196-S037 MYD88 N N CD3D N Y E009/E014-T055-S186-S052 MPLY N CD79A N Y E009/E014-T060-S175-S053 LEPR Y N CD79B N YE009/E014-T070-S085-S212 IFNGR2 Y N TNFRSF8 Y N E008/E013-T026-S054-S213CRLF2 Y N TNFRSF9 Y N E009/E014-T007-S120-S053 IL7R Y N CD79B N YE007/E012-T045-S186-S211 MPL Y N TNFRSF4 Y N E008/E013-T073-S186-X002MPL Y N N/A N/A N/A E008/E013-T074-S186-X002 MPL Y N N/A N/A N/AE007/E012-T055-S186-S053 MPL Y N CD79B N Y E008/E013-T036-S186-S053 MPLY N CD79B N Y E007/E012-T017-S058-S053 CSF2RA Y N CD79B N YE008/E013-T030-S189-S080 MYD88 N N ICOS N N E006/E011-T029-S081-S047IFNAR1 Y N CD27 Y N E009/E014-T044-S194-S050 MYD88 N N CD40 Y NE006/E011-T028-S121-X002 IL7R Y N N/A N/A N/A E008/E013-T028-S186-S053MPL Y N CD79B N Y E009/E014-T078-S142-S213 IL13RA2 Y N TNFRSF9 Y NE009/E014-T041-S186-S051 MPL Y N CD40 Y N E008/E013-T006-S186-S050 MPL YN CD40 Y N E006/E011-T028-S186-S075 MPL Y N FCGR2C Y YE006/E011-T040-S120-S038 IL7R Y N CD3E N Y E007/E012-T044-S115-S211IL5RA Y N TNFRSF4 Y N E009/E014-T039-S176-S075 LEPR Y N FCGR2C Y YE007/E012-T028-S186-S050 MPL Y N CD40 Y N E008/E013-T031-S202-S050 PRLRY N CD40 Y N E007/E012-T072-S192-S053 MYD88 N N CD79B N YE006/E011-T065-X001-S051 N/A N/A N/A CD40 Y N E007/E012-T030-S062-X002CSF3R Y N N/A N/A N/A E007/E012-T073-S186-X002 MPL Y N N/A N/A N/AE009/E014-T056-S186-S053 MPL Y N CD79B N Y E008/E013-T046-S137-X002IL12RB1 Y N N/A N/A N/A E006/E011-T016-S136-S076 IL12RB1 Y N FCGR2A Y YE007/E012-T032-S142-S037 IL13RA2 Y N CD3D N Y E007/E012-T065-S120-S215IL7R Y N TNFRSF18 Y N E009/E014-T077-S186-S047 MPL Y N CD27 Y NE009/E014-T001-S126-S051 IL9R Y N CD40 Y N E006/E011-T030-S121-S039 IL7RY N CD3G N Y E008/E013-T006-S176-S213 LEPR Y N TNFRSF9 Y NE009/E014-T032-S130-S215 IL10RB Y N TNFRSF18 Y NE008/E013-T041-S186-S039 MPL Y N CD3G N Y E009/E014-T021-S186-S047 MPL YN CD27 Y N E008/E013-T026-S137-S214 IL12RB1 Y N TNFRSF14 Y NE007/E012-T029-S116-S075 IL6R Y N FCGR2C Y Y E008/E013-T026-S106-S049IL2RG Y N CD28 N N E007/E012-T032-S168-S075 IL27RA Y N FCGR2C Y YE006/E011-T031-S186-S052 MPL Y N CD79A N Y E007/E012-T043-S186-S051 MPLY N CD40 Y N E008/E013-T066-S130-S075 IL10RB Y N FCGR2C Y YE009/E014-T019-S143-S074 IL15RA Y N FCER1G Y Y E006/E011-T010-S130-X002IL10RB Y N N/A N/A N/A E009/E014-T065-S083-S215 IFNAR2 Y N TNFRSF18 Y NE007/E012-T041-S130-S075 IL10RB Y N FCGR2C Y Y E007/E012-T044-S199-S053OSMR Y N CD79B N Y E007/E012-T075-S059-X002 CSF2RA Y N N/A N/A N/AE008/E013-T015-S170-S214 IL31RA Y N TNFRSF14 Y NE006/E011-T030-X001-S076 N/A N/A N/A FCGR2A Y Y E009/E014-T072-S186-X002MPL Y N N/A N/A N/A E008/E013-T032-S176-S211 LEPR Y N TNFRSF4 Y NE009/E014-T075-S136-S052 IL12RB1 Y N CD79A N Y E007/E012-T041-S186-S052MPL Y N CD79A N Y E008/E013-T028-S186-S039 MPL Y N CD3G N YE008/E013-T062-S186-X002 MPL Y N N/A N/A N/A E007/E012-T044-S186-S051MPL Y N CD40 Y N E007/E012-T014-S058-X002 CSF2RA Y N N/A N/A N/AE008/E013-T001-S186-S075 MPL Y N FCGR2C Y Y E009/E014-T029-S137-X002IL12RB1 Y N N/A N/A N/A E006/E011-T004-S194-S038 MYD88 N N CD3E N YE007/E012-T016-S186-X002 MPL Y N N/A N/A N/A E009/E014-T031-S186-S075MPL Y N FCGR2C Y Y E009/E014-T069-S137-S039 IL12RB1 Y N CD3G N YE007/E012-T049-S143-S216 IL15RA Y N TNFRSF18 Y NE007/E012-T028-S186-S053 MPL Y N CD79B N Y E008/E013-T011-S186-S037 MPLY N CD3D N Y E009/E014-T031-S186-S039 MPL Y N CD3G N YE009/E014-T020-S186-S050 MPL Y N CD40 Y N E008/E013-T016-S186-X002 MPL YN N/A N/A N/A E009/E014-T046-S186-S053 MPL Y N CD79B N Y

TABLE 23 Constructs with Particularly Noteworthy Enrichments inLibraries 4B and 4.1B. P3 P4 Contains Contains Cytokine ITAM CytokineITAM BlockSequence Gene receptor? motif? Gene receptor? motif?E007/E012-T078-S154-S047 IL18R1 Y N CD27 Y N E008/E013-T062-S186-X002MPL Y N N/A N/A N/A E008/E013-T055-S186-S050 MPL Y N CD40 Y NE009/E014-T057-S186-S050 MPL Y N CD40 Y N E007/E012-T077-S054-S053 CRLF2Y N CD79B N Y E007/E012-T034-S135-S211 IL11RA Y N TNFRSF4 Y NE009/E014-T071-X001-S216 N/A N/A N/A TNFRSF18 Y NE009/E014-T011-S141-S037 IL13RA1 Y N CD3D N Y E008/E013-T041-S186-S037MPL Y N CD3D N Y E006/E011-T038-S106-S039 IL2RG Y N CD3G N YE006/E011-T011-S121-X002 IL7R Y N N/A N/A N/A E007/E012-T007-S085-S215IFNGR2 Y N TNFRSF18 Y N E006/E011-T041-S186-S050 MPL Y N CD40 Y NE007/E012-T008-S064-S051 CSF3R Y N CD40 Y N E006/E011-T041-S186-S047 MPLY N CD27 Y N E008/E013-T045-S186-S051 MPL Y N CD40 Y NE008/E013-T003-S104-S216 IL2RA Y N TNFRSF18 Y N E006/E011-T019-S186-S053MPL Y N CD79B N Y E008/E013-T071-S064-S080 CSF3R Y N ICOS N NE006/E011-T021-S054-X002 CRLF2 Y N N/A N/A N/A E006/E011-T003-S135-X002IL11RA Y N N/A N/A N/A E009/E014-T020-S199-S213 OSMR Y N TNFRSF9 Y NE008/E013-T027-S121-S211 IL7R Y N TNFRSF4 Y N E009/E014-T032-S195-X002MYD88 N N N/A N/A N/A E009/E014-T050-S171-X002 IL31RA Y N N/A N/A N/AE008/E013-T069-S083-X002 IFNAR2 Y N N/A N/A N/A E008/E013-T026-S116-S038IL6R Y N CD3E N Y E009/E014-T072-S195-X002 MYD88 N N N/A N/A N/AE007/E012-T047-S058-S211 CSF2RA Y N TNFRSF4 Y N E008/E013-T046-S142-S080IL13RA2 Y N ICOS N N E006/E011-T065-S186-S076 MPL Y N FCGR2A Y YE006/E011-T062-S069-X002 EPOR Y N N/A N/A N/A E007/E012-T047-S098-X002IL1R1 Y N N/A N/A N/A E009/E014-T069-S099-S048 IL1R1 Y N CD28 N NE008/E013-T039-S141-S050 IL13RA1 Y N CD40 Y N E006/E011-T052-S130-S052IL10RB Y N CD79A N Y E008/E013-T041-S186-X002 MPL Y N N/A N/A N/AE007/E012-T019-S120-X002 IL7R Y N N/A N/A N/A E008/E013-T045-S186-S053MPL Y N CD79B N Y E006/E011-T003-S170-S039 IL31RA Y N CD3G N YE007/E012-T047-S058-S051 CSF2RA Y N CD40 Y N E007/E012-T069-S109-X002IL3RA Y N N/A N/A V/A E009/E014-T019-S130-S075 IL10RB Y N FCGR2C Y YE006/E011-T047-S054-S053 CRLF2 Y N CD79B N Y

What is claimed is:
 1. A method for isolating a microenvironmentrestricted antigen-specific targeting region (ASTR), comprising panninga polypeptide library by: a) contacting the polypeptide library underaberrant conditions with a target antigen bound to a solid support,wherein clones expressing polypeptides that bind the target antigenremain bound to the solid support through the target antigen; b)incubating the solid supports with bound polypeptides underphysiological conditions; and c) collecting clones that elute from thesolid support under the physiological conditions, thereby isolating themicroenvironment restricted antigen-specific targeting region.
 2. Themethod of claim 1, wherein the polypeptide library is a phage displaylibrary, and wherein the collected clones are collected phage clones. 3.The method of claim 2, wherein the method further comprises infectingbacterial cells with the collected phage clones to generate a refinedphage display library.
 4. The method of claim 3, wherein the refinedphage display library is used as the polypeptide library in a subsequentround of panning a polypeptide library, and wherein the contacting,incubating, and collecting steps are repeated for 1 to 1000 cycles,using the refined phage display library generated from the previouscycle.
 5. The method of claim 1, wherein the contacting, incubating, andcollecting steps are repeated for 1 to 1000 cycles.
 6. The method ofclaim 5, wherein the polypeptides in the polypeptide library are notmutated or evolved before the panning or between rounds of panning. 7.The method of claim 1, wherein one or more polypeptides in thepolypeptide library or one or more polynucleotides encoding polypeptidesin the polypeptide library are subjected to mutagenesis before thecontacting step.
 8. The method of claim 1, wherein the physiologicalconditions comprise temperature, pH, osmotic pressure, osmolality,oxidative stress, and/or electrolyte concentration.
 9. The method ofclaim 1, wherein microenvironment restricted ASTR binds to its cognateantigen with a 10% greater affinity under aberrant conditions thanphysiological conditions.
 10. The method of claim 1, further comprisinggenerating a polynucleotide that encodes a polypeptide comprising themicroenvironment restricted ASTR, a transmembrane domain, and anintracellular activating domain.
 11. The method of claim 10, wherein theASTR is an scFv fragment comprising a heavy chain variable region and alight chain variable region.
 12. The method of claim 10, wherein theASTR is an scFv-Fc antibody.
 13. The method of claim 1, wherein themicroenvironment restricted ASTR is an antibody, an antigen, a ligand, areceptor binding domain of a ligand, a receptor, a ligand binding domainof a receptor, or an affibody.
 14. The method of claim 1, wherein themicroenvironment restricted ASTR is a full-length antibody, asingle-chain antibody, a single-chain variable fragment, an Fabfragment, an Fab′ fragment, an (Fab′)2 fragment, an Fv fragment, adivalent single-chain antibody, or a diabody.
 15. The method of claim 1,wherein the solid supports comprise glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylose, natural cellulose, modifiedcellulose, polyacrylamide, agarose, and/or magnetic solid supports. 16.The method of claim 1, wherein the solid supports comprise one or morebeads, particles, and/or microspheres, and/or comprise a surface of atube or plate or a filter membrane.
 17. The method of claim 1, whereinthe method comprises a cell-based assay comprising cells.
 18. The methodof claim 17, wherein the cells comprise a polynucleotide encoding amicroenvironment restricted ASTR, wherein the nucleotide sequence of thepolynucleotide encoding the isolated microenvironment restricted ASTR isdetermined by sequencing nucleotides of a cell expressing themicroenvironment restricted ASTR.
 19. A method for performing adoptivecell therapy on a subject, comprising: a) contacting a T cell and/or anNK cell from the subject ex vivo with replication incompetentrecombinant retroviral particles, wherein the replication incompetentrecombinant retroviral particles comprise: i. a polynucleotidecomprising one or more transcriptional units, wherein each of the one ormore transcriptional units is operatively linked to a promoter active inT and/or NK cells, and wherein the one or more transcriptional unitsencode a first engineered polypeptide; and ii. an activation element onthe surfaces of the replication incompetent recombinant retroviralparticles, wherein the activation element is fused to a membraneattachment sequence, wherein said contacting facilitates transduction ofthe T cell and/or NK cell; b) expanding the transduced T cell and/or NKcell ex vivo for fewer than 4 cell divisions; and c) introducing theexpanded T cells and/or NK cells into the subject, thereby performingadoptive cell therapy on the subject.
 20. A method of introducing anexogenous nucleic acid into a T cell and/or an NK cell, comprisingcontacting the T cell and/or NK cell ex vivo, with replicationincompetent recombinant retroviral particles, wherein the replicationincompetent recombinant retroviral particles comprise: a) the exogenousnucleic acid, wherein the exogenous nucleic acid comprises apolynucleotide comprising one or more transcriptional units, whereineach of the one or more transcriptional units is operatively linked to apromoter active in T and/or NK cells, and wherein the one or moretranscriptional units encode a first engineered polypeptide; and b) anactivation element on the surfaces of the replication incompetentrecombinant retroviral particles, wherein the activation element isfused to a membrane attachment sequence, wherein the T cell and/or NKcell is contacted ex vivo with the replication incompetent recombinantretroviral particles for less than 14 hours, and wherein after saidcontacting the T cell and/or NK cell comprises the exogenous nucleicacid.